{"Affiliation":[{"label":"Affiliation","value":"Applied Science, Faculty of","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."},{"label":"Affiliation","value":"Arts and Sciences, Irving K. Barber School of (Okanagan)","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."},{"label":"Affiliation","value":"Chemistry, Department of (Okanagan)","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."},{"label":"Affiliation","value":"Engineering, School of (Okanagan)","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."}],"AggregatedSourceRepository":[{"label":"Aggregated Source Repository","value":"DSpace","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","classmap":"ore:Aggregation","property":"edm:dataProvider"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","explain":"A Europeana Data Model Property; The name or identifier of the organization who contributes data indirectly to an aggregation service (e.g. Europeana)"}],"Citation":[{"label":"Citation","value":"AMB Express. 2018 Nov 03;8(1):181","attrs":{"lang":"en","ns":"https:\/\/open.library.ubc.ca\/terms#identifierCitation","classmap":"oc:PublicationDescription","property":"oc:identifierCitation"},"iri":"https:\/\/open.library.ubc.ca\/terms#identifierCitation","explain":"UBC Open Collections Metadata Components; Local Field; Indicates a bibliographic reference for the resource if it has been previously published."}],"CopyrightHolder":[{"label":"Copyright Holder","value":"The Author(s)","attrs":{"lang":"en","ns":"https:\/\/open.library.ubc.ca\/terms#rightsCopyright","classmap":"oc:PublicationDescription","property":"oc:rightsCopyright"},"iri":"https:\/\/open.library.ubc.ca\/terms#rightsCopyright","explain":"UBC Open Collections Metadata Components; Local Field; Refers to the publisher or author who holds the copyright."}],"Creator":[{"label":"Creator","value":"Fordwour, Osei B","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/creator","classmap":"dpla:SourceResource","property":"dcterms:creator"},"iri":"http:\/\/purl.org\/dc\/terms\/creator","explain":"A Dublin Core Terms Property; An entity primarily responsible for making the resource.; Examples of a Contributor include a person, an organization, or a service."},{"label":"Creator","value":"Luka, George","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/creator","classmap":"dpla:SourceResource","property":"dcterms:creator"},"iri":"http:\/\/purl.org\/dc\/terms\/creator","explain":"A Dublin Core Terms Property; An entity primarily responsible for making the resource.; Examples of a Contributor include a person, an organization, or a service."},{"label":"Creator","value":"Hoorfar, Mina","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/creator","classmap":"dpla:SourceResource","property":"dcterms:creator"},"iri":"http:\/\/purl.org\/dc\/terms\/creator","explain":"A Dublin Core Terms Property; An entity primarily responsible for making the resource.; Examples of a Contributor include a person, an organization, or a service."},{"label":"Creator","value":"Wolthers, Kirsten R","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/creator","classmap":"dpla:SourceResource","property":"dcterms:creator"},"iri":"http:\/\/purl.org\/dc\/terms\/creator","explain":"A Dublin Core Terms Property; An entity primarily responsible for making the resource.; Examples of a Contributor include a person, an organization, or a service."}],"DateAvailable":[{"label":"Date Available","value":"2018-11-05T18:24:12Z","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/issued","classmap":"edm:WebResource","property":"dcterms:issued"},"iri":"http:\/\/purl.org\/dc\/terms\/issued","explain":"A Dublin Core Terms Property; Date of formal issuance (e.g., publication) of the resource."}],"DateIssued":[{"label":"Date Issued","value":"2018-11-03","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/issued","classmap":"oc:SourceResource","property":"dcterms:issued"},"iri":"http:\/\/purl.org\/dc\/terms\/issued","explain":"A Dublin Core Terms Property; Date of formal issuance (e.g., publication) of the resource."}],"Description":[{"label":"Description","value":"Acetone monooxygenase (ACMO) is a unique member of the Baeyer\u2013Villiger monooxygenase (BVMO) family based on its ability to act on small ketones, such as acetone. Herein, we performed a kinetic analysis of ACMO from the propane-utilizing bacterium Gordonia sp. strain TY-5 to assess its preference for smaller ketone substrates. Steady state kinetic analysis of ACMO with a range of linear (C3\u2013C7) and cyclic ketones (C4\u2013C6) reveals that the enzyme elicits the highest catalytic efficiency towards butanone and cyclobutanone. Stopped-flow and inhibition studies further revealed that ACMO has a relatively weak binding affinity for the coenzyme with a dissociation constant of 120\u00a0\u03bcM. We show through mutagenesis that sequence variation in the residue that coordinates to the 2\u2032-phosphate of NADP(H) partially accounts for the weaker binding affinity observed. As for shown for related BVMOs, NADP+ stabilizes the C4a-peroxyflavin intermediate in ACMO; however, the rate of its formation is considerably slower in ACMO. The observed rate constant for NADPH-dependent flavin reduction was 60\u00a0s\u22121 at 25\u00a0\u00b0C, and experiments performed with 4(R)-[4-2H]NADPH confirm that the C4-pro-R-hydride from the nicotinamide ring is transferred to the FAD. The latter experimental result suggests that the nicotinamide ring rotates within the active site to carry out its two functional roles: reduction of the FAD cofactor and stabilization of the C4a-peroxyflavin adduct.","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/description","classmap":"dpla:SourceResource","property":"dcterms:description"},"iri":"http:\/\/purl.org\/dc\/terms\/description","explain":"A Dublin Core Terms Property; An account of the resource.; Description may include but is not limited to: an abstract, a table of contents, a graphical representation, or a free-text account of the resource."}],"DigitalResourceOriginalRecord":[{"label":"Digital Resource Original Record","value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/67757?expand=metadata","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","classmap":"ore:Aggregation","property":"edm:aggregatedCHO"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","explain":"A Europeana Data Model Property; The identifier of the source object, e.g. the Mona Lisa itself. This could be a full linked open date URI or an internal identifier"}],"FullText":[{"label":"Full Text","value":"Fordwour et al. AMB Expr           (2018) 8:181  https:\/\/doi.org\/10.1186\/s13568-018-0709-xORIGINAL ARTICLEKinetic characterization of acetone monooxygenase from Gordonia sp. strain TY-5Osei Boakye Fordwour1, George Luka2, Mina Hoorfar2 and Kirsten R. Wolthers1*Abstract Acetone monooxygenase (ACMO) is a unique member of the Baeyer\u2013Villiger monooxygenase (BVMO) family based on its ability to act on small ketones, such as acetone. Herein, we performed a kinetic analysis of ACMO from the propane-utilizing bacterium Gordonia sp. strain TY-5 to assess its preference for smaller ketone substrates. Steady state kinetic analysis of ACMO with a range of linear (C3\u2013C7) and cyclic ketones (C4\u2013C6) reveals that the enzyme elicits the highest catalytic efficiency towards butanone and cyclobutanone. Stopped-flow and inhibition studies further revealed that ACMO has a relatively weak binding affinity for the coenzyme with a dissociation constant of 120 \u03bcM. We show through mutagenesis that sequence variation in the residue that coordinates to the 2\u2032-phosphate of NADP(H) partially accounts for the weaker binding affinity observed. As for shown for related BVMOs,  NADP+ stabi-lizes the C4a-peroxyflavin intermediate in ACMO; however, the rate of its formation is considerably slower in ACMO. The observed rate constant for NADPH-dependent flavin reduction was 60 s\u22121 at 25 \u00b0C, and experiments performed with 4(R)-[4-2H]NADPH confirm that the C4-pro-R-hydride from the nicotinamide ring is transferred to the FAD. The lat-ter experimental result suggests that the nicotinamide ring rotates within the active site to carry out its two functional roles: reduction of the FAD cofactor and stabilization of the C4a-peroxyflavin adduct.Keywords: Bayer\u2013Villiger monooxygenase, Acetone monooxygenase, Stopped-flow spectroscopy\u00a9 The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:\/\/creat iveco mmons .org\/licen ses\/by\/4.0\/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.IntroductionBaeyer\u2013Villiger monooxygenases (BVMOs) are flavin-containing enzymes that oxidize ketones to esters or lac-tones, using  O2 and reducing equivalents from NAD(P)H. They hold promise as versatile biocatalysts given their broad substrate scope and excellent enantio-, and regi-oselectivity (Leisch et\u00a0 al. 2011; Torres Pazmino et\u00a0 al. 2010). In addition to Baeyer\u2013Villiger oxidations, BVMOs can also oxidize a range of other functional groups including aldehydes, sulfides, amines, phosphines, and selenides and iodide containing molecules (de Gonzalo et\u00a0 al. 2010; Balke et\u00a0 al. 2012; Rehdorf et\u00a0 al. 2009). The majority of BVMOs are type I BVMOs, which bind FAD, elicit a high preference for NADPH, and are composed of a single polypeptide. The substrate scope and catalytic promiscuity varies greatly among the enzyme family. For example, some BVMOs act on bulky steroids, sesquiter-penes or aflatoxins, while the substrate scope of others is limited to smaller (a)cyclic ketones (Kolek et\u00a0al. 2008).The BVMO catalytic mechanism was formulated from kinetic and spectroscopic analysis of cyclohex-anone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871, one of the more comprehensively studied members of the enzyme family (Scheme\u00a01) (Ryerson et\u00a0al. 1982; Sheng et\u00a0al. 2001). Following reduction of oxidized FAD by NADPH, the resulting  FADH\u2212 reacts with  O2 to form the C4a-peroxyflavin adduct. In BVMO, the pro-longed lifetime of this intermediate is partially attributed to  NADP+, which remains situated in the active site and is the final product to dissociate from the enzyme. If the enzyme undergoes productive catalysis (i.e. performs a monooxygenation reaction), then the C4a-peroxyflavin adduct attacks the carbonyl group of the ketone sub-strate, leading to formation of a tetrahedral Criegee intermediate which then collapses to form the lactone and hydroxyflavin intermediate. Release of water from hydroxyflavin returns the FAD cofactor to its oxidized Open Access*Correspondence:  kirsten.wolthers@ubc.ca 1 Department of Chemistry, University at the British Columbia, Okanagan Campus, 3247 University Way, Kelowna, BC V1V 1V7, CanadaFull list of author information is available at the end of the articlePage 2 of 13Fordwour et al. AMB Expr           (2018) 8:181 state for another catalytic cycle. In the absence of sub-strate, the C4a-peroxyflavin adduct can become proto-nated and collapse to form  H2O2, leading to uncoupled NADPH oxidation.Acetone monooxygenase (ACMO) is an example of a Type I BVMO that functions in the catabolism of small organic ketones. The enzyme was initially isolated from Gordonia sp. strain TY-5, a Gram-positive bacterium capable of aerobic growth with gaseous propane as the sole carbon source (Hausinger 2007). The bacterium encodes an NADH-dependent dinuclear-iron-containing multicomponent monooxygenase that converts propane to 2-propanol and three secondary alcohol dehydro-genases that oxidize 2-propanol to acetone. ACMO, encoded by the acmA gene is part of a bicistronic operon that also includes the acmB gene. ACMO was shown to convert acetone to methyl acetate, while the gene prod-uct of acmB, an esterase, hydrolyzes methyl acetate to methanol and acetate (Kotani et\u00a0al. 2007).ACMO appears to be unique among BVMOs based on its ability to act on acetone. More well studied BVMO family members such as CHMO and phenylacetone monooxygenase from Thermobifida fusca (PAMO) are not able to catalyze the oxidation of this smallest ketone (Fraaije et\u00a0 al. 2005; Donoghue et\u00a0 al. 1976). Our group was interested in examining AMCO\u2019s catalytic efficiency towards acetone to assess its potential to be used as a biosensor, for example in the detection of ketone bod-ies in the salvia of diabetics. Herein, we measured the enzyme\u2019s catalytic efficiency for acetone, relative to larger linear and cyclic ketones. The stability of the enzyme at various temperatures was also measured along with the pH-dependence of its activity. Stopped-flow analysis of the reductive half reaction with 4(R)-[4-2H]NADPH sup-ports transfer of the proR-hydrogen, while pre-steady state kinetic analysis of the oxidative half reaction reveals that the C4a-peroxyflavin intermediate is a less stable intermediate in ACMO. Finally molecular modeling revealed structural variation in the coenzyme binding pocket that results in weaker NADP(H) binding affinity.Materials and methodsMaterialsNADPH,  NADP+, ketone substrates, xanthine, xanthine oxidase, methyl viologen, and benzyl viologen were pur-chased from Sigma. Restriction enzymes were purchased from New England Biolabs, and PfuTurbo DNA polymer-ase was obtained from Agilent Technologies. All other chemicals and media were purchased from VWR. 4(R)-[4-2H]NADPH was synthesized and purified following a published protocol (Bowman et\u00a0al. 2010).Construction of the ACMO expression vectorThe cDNA encoding ACMO was synthesized by Gen-Script (Piscataway, NJ, USA; GenBank Accession num-ber MH880286).). The coding sequence was optimized for expression in E. coli and subcloned into pET23a(+), which resulted in a C-terminal hexahistidine tag. This construct did not result in the overexpression of a solu-ble form of ACMO, so the opportunity was taken to sub-clone the ACMO cDNA into the pGEX4T1 vector, which places a glutathione S-transferase (GST) motif onto the N-terminus of the protein. The forward (5\u2032-CGG GAT CCA TGA GCA CGA CGA CGC TGG-3\u2032) and reverse (5\u2032-CCG CTC GAG TTA CGA CAG TGC GAA ACC-3\u2032) primers used to amplify the ACMO coding sequence har-bored BamHI and XhoI restriction enzyme sites (in bold), respectively. The cDNA for ACMO was PCR ampli-fied using PfuTurbo DNA polymerase, digested with BamHI and XhoI and ligated into pGEX4T1 also cut with the same restriction enzymes. The resulting construct (pGEX-ACMO) was transformed into E. coli DH5\u03b1 for protein expression.Construction of ACMO H325K and A443\u0394 variantsThe H325K and A443\u0394 variants were constructed using the QuikChange II site-directed mutagenesis kit from Agilent Technologies using the pGEX-ACMO vector as a template. The forward and reverse primers used for the H325K substitution are as follows: 5\u2032-GAT TAC GGC TTC GGT ACC AAA CGC GTG CCG CTG GAA AC-3\u2032 and 5\u2032-GAT TAC GGC TTC GGT ACC TTT CGC GTG CCG CTG GAA AC-3\u2032. The oligonucleotides for the deletion of A433 are 5\u2032-CCG CTG GCT CCG AGC GCT CTG TCG AAT ATG-3\u2032 and 5\u2032-CAT ATT GCA CAG AGC GCT CGG AGC CAG CGG-3\u2032. The success-ful incorporation of lysine at position 325, the deletion of A433, and the absence of any unintended sequence alter-ations was confirmed by DNA sequencing at the NAPS Scheme 1 Catalytic mechanism of ACMOPage 3 of 13Fordwour et al. AMB Expr           (2018) 8:181 DNA Sequencing Laboratory at University of British Columbia, Vancouver, Canada.Expression and purification of ACMO and the H325K and A443\u0394 variantsAn overnight culture of DH5\u03b1 harbouring the pGEX-ACMO plasmid was used to inoculate six 1.5 L flasks of Terrific Broth supplemented with ampicillin (100\u00a0\u03bcg\/mL). Cultures were grown at 30\u00a0 \u00b0C with agitation, set at 200\u00a0 rpm, to an  OD600 of 0.8. Recombinant protein expression was induced with the addition of 0.2\u00a0mM of IPTG to the cell cultures. Following continued growth at 25\u00a0\u00b0C for 16\u00a0h, the cells were harvested by centrifugation (6000\u00d7g for 15\u00a0min). The cell pellet was frozen at \u2212\u00a080\u00a0\u00b0C until purification.All purification steps were performed on ice or at 4\u00a0\u00b0C. The frozen cell pellet (~ 25\u00a0g) was resuspended in 200\u00a0mL of GST bind\/wash buffer (10\u00a0 mM  Na2HPO4, 1.8\u00a0 mM  KH2PO4, 0.14\u00a0M NaCl, 2.7\u00a0mM KCl, 1\u00a0mM EDTA, 1\u00a0mM DTT, pH 7.5) containing 10\u00a0\u03bcg\/mL of benzamidine and 1\u00a0 mM phenylmethylsulfonyl fluoride. The cells were lysed and the genomic DNA was sheared by sonication and then the cell suspension was centrifuged (38,000\u00d7g for 60\u00a0 min). The clarified cell extract was applied to a 5.5 \u00d7 4.0\u00a0 cm column containing glutathione Sepha-rose 4B (GE Healthcare Life Sciences) equilibrated with GST bind\/wash buffer. The column was washed with 1 L (~ 10 column volumes) of GST bind\/wash buffer. The GSTACMO chimera was eluted with 50\u00a0 mM Tris\u2013HCl, pH 8.0 supplemented with 1\u00a0 mM dithiothreitol and 10\u00a0mM glutathione. The eluate was dialyzed against 4 L of GST bind\/wash buffer containing 1\u00a0 mM EDTA and 1\u00a0mM dithiothreitol for 16\u00a0h with thrombin to cleave the GST tag. To separate ACMO from the uncut GSTACMO chimera and thrombin, the dialyzed protein was applied to a glutathione Sepharose 4B (2.5 \u00d7 3.0\u00a0 cm) column equilibrated with GST bind\/wash buffer. The column was then washed with 50\u00a0 mL of the GST bind\/wash buffer with 10% glycerol (v\/v). The yellow fractions were col-lected and the protein was concentrated using a cen-trifugal concentrator with a 30\u00a0kDa cutoff filter. ACMO was then applied to a Q-Sepharose column (2.6 \u00d7 14\u00a0cm) equilibrated with buffer A (50\u00a0mM Tris\u2013HCl pH 7.5, 10% glycerol). The protein was eluted with a linear gradient to 50\u00a0mM Tris\u2013HCl pH 7.5, 10% glycerol, 0.5\u00a0M NaCl. The eluted protein was concentrated and stored at \u2212\u00a080\u00a0\u00b0C in 20% glycerol (v\/v).Determining the molar absorption coefficient of ACMOThe UV\u2013visible absorbance spectrum of ACMO was recorded between 700 and 200\u00a0nm in 50\u00a0mM HEPES pH 7.5 at 25\u00a0 \u00b0C. The flavoprotein (1.2\u00a0 mL) was then added to 0.3\u00a0 mL of a 10% solution of SDS to obtain a final concentration of 0.2% SDS. Changes in the spectra of the flavin cofactor were recorded at 4\u00a0 min intervals until a constant absorbance spectrum was obtained. The protein solution was then centrifuged at 14,000\u00a0rpm for 10\u00a0min in a microcentrifuge and the spectrum of the FAD liberated from the protein was measured. The concentration of free FAD released from the protein was calculated using a molar absorptivity of free FAD (\u0190450 = 11,300\u00a0M\u22121\u00a0cm\u22121). The molar absorption coefficient of ACMO was calcu-lated to be \u0190443 = 12,200\u00a0 M\u22121\u00a0 cm\u22121 after correcting for the dilution of the protein.NADP+ binding constant determinationUV\u2013visible absorbance spectroscopy was used to deter-mine the equilibrium dissociation constant for  NADP+. The assays were performed on a Perkin Elmer Lambda 25 UV\u2013visible spectrophotometer in 50\u00a0mM HEPES pH 7.5 at 25\u00a0 \u00b0C.  NADP+ was sequentially added to an absorb-ance cuvette containing 50\u00a0\u00b5M of flavoprotein, achieving  NADP+ concentrations ranging from 0 to 200\u00a0 \u03bcM. The change in absorbance at a given wavelength was fit to the quadratic binding isotherm (Eq.\u00a01).where  Lo is total  NADP+ concentration; Eo is total enzyme concentration; \u0394A is the change in absorbance, \u0394Amax is the maximum change in absorbance; and Kd is the dissociation constant for the GSTACMO-NADP+ complex.Determining the reduction potential of GSTACMOThe reduction potential of GSTACMO was determined by Massey\u2019s method (Massey 1991). The method relies on the near simultaneous reduction of GSTACMO and a ref-erence dye with a known reduction potential by reducing equivalents derived from xanthine oxidase and xanthine. The reference dye used in the GSTACMO reduction was indigo disulfonate, which has a reduction potential of Em0 = \u2212\u00a0 116\u00a0 mV at pH 7.0. The titration of GSTACMO was performed at 25\u00a0\u00b0C in a total volume of 1\u00a0mL in an anaerobically maintained glove box in 50\u00a0 mM HEPES, pH 7.0, that was made anaerobic by the same method described for the stopped-flow experiments. A concen-trated 2\u00a0 mL stock of GSTACMO (36\u00a0 \u03bcM) was intro-duced into the glove box and gel filtered over a 10\u00a0 mL PD10 column (Bio-Rad) equilibrated with anaerobic buffer (50\u00a0mM HEPES, pH 7.0). Stock solutions of ben-zyl viologen, indigo disulfonate, xanthine and xanthine oxidase were prepared by dissolving lyophilized powders of each in anaerobic buffer in the glove box. The reaction contained 20\u00a0\u00b5M GSTACMO, 20\u00a0\u00b5M indigo disulfonate, (1)\ufffdA =(\ufffdAmax2Eo){Eo + Lo + Kd \u2212[(Eo + LO + Kd)2\u2212 4EoLo]1\/2}Page 4 of 13Fordwour et al. AMB Expr           (2018) 8:181 0.5\u00a0 mM xanthine, and 5\u00a0 \u00b5M benzyl viologen. Following the addition of 0.25\u00a0 \u00b5M xanthine oxidase, the reaction mixture was placed in a Lambda 265 spectrophotometer (Perkin Elmer) also housed in the glove box, and reduc-tion of the enzyme and dye was monitored over 2\u00a0 h by recording the absorption spectrum of the reaction mix-ture every minute. The concentrations of the oxidized and reduced enzyme (Eox and Ered) throughout the titra-tion were determined at 458\u00a0nm (the isosbestic point of indigo disulfonate), while the concentration of the dye (both the oxidized and reduced forms; Dox and Dred) was determined at 610\u00a0nm, where oxidized and hydroquinone forms of the FAD cofactor do not absorb. The reduction potential of the enzyme (Ee) and the dye (Dd) were calcu-lated from the following equations:where n is the number of electrons. At equilibrium, Ed is equal to Ee and Eqs.\u00a01 and 2 can be rearranged to the fol-lowing equation:Equation\u00a0 3 enables the reduction potential of GSTACMO, E\u25e6e to be determined from the y-intercept of a plot of log(Ered\/Eox) versus log(Dred\/Dox).Steady\u2011state kinetic assaysThe steady-state parameters of ACMO and GSTACMO were determined for a variety of linear and cyclic ketones by following the rate of oxidation of NADPH at 340\u00a0nm (\u0394\u0190340 = 6.22\u00a0 mM\u22121  cm\u22121). The reactions were per-formed in a total volume of 1\u00a0mL in air-saturated 50\u00a0mM HEPES buffer, pH 7.5 at 25\u00a0 \u00b0C using a Lambda 25 UV\u2013visible spectrophotometer (Perkin Elmer) placed on the laboratory bench. The steady-state kinetic parameters were determined by measuring the initial velocity for the oxidation\u00a0 of\u00a0 various ketones in 1\u00a0 mL reaction mixtures that contained 100\u00a0\u00b5M NADPH and variable concentra-tions (0.1\u20135\u00a0 mM) of the ketone substrate. The Michae-lis constant for NADPH was determined with variable concentrations of NADPH and a fixed concentration of butanone (200\u00a0 \u03bcM). All steady-state reactions were ini-tiated with 23\u201345\u00a0 nM of ACMO or GSTACMO. The initial velocities were plotted as a function of substrate (2)Ee = E\u25e6e \u22120.0592nelog(EredEox)(3)Ed = E\u25e6d\u22120.0592ndlog(DredDox)(4)log(EredEox)=ne(E\u25e6e \u2212 E\u25e6d)0.0592+(nend)log(DredDox)concentration and fitted to the Michealis Menten equa-tion using nonlinear least squares analysis with the computer program Origin 8.0 (OriginLab). The  NADP+ inhibition assays were performed with variable  NADP+ (0\u20131\u00a0mM) and NADPH (0.2\u2013100\u00a0\u03bcM) concentrations in the presence of 200\u00a0 \u03bcM butanone. The inhibition data were fitted to the competitive inhibition equation using Origin 8.0. The rate of uncoupled NADPH oxidation was determined by measuring absorbance changes at 340\u00a0nm in the presence of 100\u00a0\u03bcM NADPH and in the absence of a ketone substrate.Stability assays and pH\u2011dependenceThe thermostability of GSTACMO was analyzed by incu-bating the purified enzyme (0.5\u00a0 \u03bcM) at a set tempera-ture (ranging from 5 to 45\u00a0\u00b0C) for 1\u00a0h in 50\u00a0mM HEPES, pH 7.5. After incubation the samples were placed on ice and then measured for activity by following the rate of NADPH oxidation spectrophotometrically at 340\u00a0 nm. Residual activity was measured at 25\u00a0 \u00b0C in 50\u00a0 mM HEPES, pH 7.5 containing 100\u00a0\u03bcM NADPH and 200\u00a0\u03bcM butanone. The reactions were initiated with the addi-tion of 25\u00a0 nM GSTACMO. The activity of GSTACMO was measured at a range of pH values (5.5\u20139.0) using a three-component buffer containing 50\u00a0 mM each of MES, HEPES and CHES. Each 1\u00a0 mL contained 100\u00a0 \u03bcM NADPH, 200\u00a0 \u03bcM butanone and 50\u00a0 nM of GSTACMO. The bell-shaped pH profiles where catalysis requires the ionization of a group with a low pKa and the protonation of a group having a higher pKa were fit to the equation:where Y is the observed velocity, YH is the velocity when both ionizable groups are in their preferred ionization state for maximal activity, and Ka1 and Ka2 are the dissoci-ation constants for the groups that ionize at low and high pH, respectively. The activity at each pH and temperature was measured in triplicate.Pre\u2011steady state kineticsPre-steady state kinetic assays were performed under anaerobic conditions using a SF-61DX2 stopped-flow apparatus (TgK Scientific). The sample-handling unit of the stopped-flow is housed in a glove box (Belle Tech-nology) with  O2 concentration < 5\u00a0 ppm. The reductive and oxidative half reactions were performed at 25\u00a0 \u00b0C in 50\u00a0mM HEPES, pH 7.5 with 20% glycerol. The buffer was made anaerobic by purging with nitrogen gas for 2\u00a0h followed by a 16\u00a0h equilibration in the glove box. Stock solutions of NADPH and  NADP+ were prepared by dis-solving lyophilized powders of the coenzyme in anaerobic (5)log Y = log[YH1+H\/Ka2 + Ka1\/H]Page 5 of 13Fordwour et al. AMB Expr           (2018) 8:181 buffer. The enzyme was diluted to the appropriate con-centration with anaerobic buffer. The reductive-half reac-tion (following NADPH-dependent reduction of oxidized GSTACMO) was monitored by rapidly mixing the oxi-dized enzyme with an equal volume of NADPH at a con-centration that was at a minimum sevenfold higher than the protein concentration so as to maintain pseudo-first order conditions. Changes in the flavin absorbance spec-tra were monitored at 443\u00a0 nm using a photomultiplier and the absorbance traces were fitted to a single expo-nential equation using Kinetic Studio (TgK Scientific). The concentration of the protein and cofactors were each diluted twofold in the observation cell. The NADPH con-centration dependence profiles were fit to the following hyperbolic equation:where kred is the limiting rate constant of flavin reduction and Kd is the dissociation constant for NADPH.The oxidative half-reaction was monitored under NADPH uncoupling conditions (in the absence of ketone substrate) and under normal turnover conditions (in the presence of butanone). In the first experiment, an anaero-bic solution of GSTACMO (20\u00a0\u03bcM) was reduced with the addition of an equimolar amount of NADPH.  NADP+ (at a final concentration of 500\u00a0 \u03bcM) was added to the pre-reduced enzyme and then this solution was rapidly mixed with an equal volume of air-saturated 50\u00a0mM HEPES pH 7.5, 20% glycerol. The second experiment was performed under the same conditions except that 400\u00a0\u03bcM butanone was added to the aerated buffer. Butanone was chosen as a substrate as it elicited the highest catalytic efficiency for ACMO. Changes in the flavin spectra were monitored at multiple wavelengths using a photodiode array detector or at individual wavelengths with a photomultipier. Typi-cally, five absorbance traces in single wavelength mode were fitted to a standard single or double exponential equation to extract the observed rate constants.ResultsPurification and spectral characterization of ACMORecombinant ACMO was expressed as a GST fusion protein, which enabled the first purification step to involve glutathione affinity chromatography. The GST-tag appeared to stabilize the flavoprotein as removal of the tag following thrombin cleavage caused the protein to slowly precipitate in 50\u00a0mM Tris\u2013HCl, pH 7.5 at 4\u00a0\u00b0C. To prevent precipitation, glycerol (10% v\/v) was added to the flavoenzyme prior to loading on to the Q-Sepharose column. Following elution from the Q-Sepharose col-umn, the protein was shown to have a molecular mass (6)kobs =kred[NADPH ]Kd + [NADPH ]of ~ 60\u00a0 kDa (close to the calculated molecular mass of 59,780\u00a0 Da) and was 90% homogeneous, as deduced from a Coomassie Blue stained SDS-PAGE gel (Addi-tional file\u00a0 1: Fig. S1). Approximately, 10\u00a0 mg of protein was obtained from 1 L of bacterial culture. The UV\u2013vis-ible absorbance spectrum of ACMO showed absorb-ance maxima at 380 and 443\u00a0 nm, typical of flavins and flavoproteins (Fig.\u00a0 1). Release of the FAD cofactor from ACMO in the presence of 0.2% SDS, enabled the extinc-tion coefficient of ACMO (\u03b5443\u00a0nm = 12,200 \u00b1 490\u00a0M\u22121\u00a0cm\u22121) to be calculated from the known extinction coefficient of free FAD.Steady state assaysThe steady state kinetic parameters of ACMO were determined in 50\u00a0mM HEPES, pH 7.5 at 25\u00a0\u00b0C for a num-ber of linear and cyclic ketones (Table\u00a0 1). The rate of NADPH oxidation (measured by the absorbance change at 340\u00a0nm) in the presence of various ketones was used to access the enzyme\u2019s substrate scope. It has been reported for PAMO and CHMO that either release of  NADP+ or dehydration of the C4a-hydroxyflavin is the rate-deter-mining step in catalysis, not oxidation of the ketone sub-strate (Sheng et\u00a0al. 2001; Torres Pazmino et\u00a0al. 2008). As a result these enzymes elicit similar turnover numbers with a broad selection of substrates (Donoghue et\u00a0 al. 1976; Mascotti et\u00a0 al. 2013, 2014). We observe a similar phe-nomenon with ACMO, as Table\u00a01 shows that the enzyme exhibits narrow range of kcat values (1.4\u20134.3\u00a0 s\u22121) for a variety of ketone substrates, indicating that like other BVMOs, oxygen insertion into the substrate is not the rate-determining step of catalysis. As shown in Table\u00a01, ACMO elicits low Km values (and correspondingly high kcat\/Km values) for small (a)cyclic ketones like butanone and cyclic butanone. The exception is acetone, which had Fig. 1 UV\u2013visible absorbance spectra of ACMO showing the diagnostic flavin peaks at 380 and 440 nm. The black line denotes the spectrum of ACMO while the dashed line is the released FAD cofactor following incubation of ACMO in 0.2% SDS for 10 min at 25 \u00b0C in 50 mM HEPES\u2013NaOH pH 7.5Page 6 of 13Fordwour et al. AMB Expr           (2018) 8:181 a 500-fold higher Km and a 700-fold lower kcat\/Km com-pared to butanone. For the acyclic ketones, lengthening of the chain or branching of the substrate with the addi-tion of methyl groups resulted in an increase in the Km, suggesting that these bulkier substrates bind more weakly to the enzyme. Likewise, a progressive increase in the size of the ring of the cyclic ketone led to a gradual increase in Km and a decrease in kcat\/Km. ACMO exhibited no activity towards cycloheptanone. However, ACMO did show activity for phenylacetone and bicyclo[3.2.0]hept-2-en-6-one.The rate of uncoupled NADPH oxidation was relatively high at 0.26\u00a0s\u22121, ~ tenfold lower than that observed in the presence of saturating amounts of the ketone substrate (Table\u00a02). By comparison, the rate of uncoupled NADPH oxidation in PAMO is 0.02\u00a0 s\u22121 (Torres Pazmino et\u00a0 al. 2008). The relatively high rate of NADPH oxidase activ-ity for ACMO suggests that the C4a-peroxyflavin is less stable in this enzyme and more prone to decay to  H2O2. The Michaelis constant (Km) for NADPH, determined in the presence of 200\u00a0 \u03bcM butanone was 6.7 \u00b1 0.8\u00a0 \u03bcM. ACMO is highly specific for NADPH as no activity was detected with NADH as the reductant.  NADP+ was found to be a poor competitive inhibitor of ACMO with a Ki of 166 \u00b1 13\u00a0\u03bcM. By comparison, PAMO and CHMO elicit Ki values of 2.7\u00a0\u03bcM and 35\u00a0\u03bcM, respectively, for the oxidized coenzyme (Ryerson et\u00a0al. 1982; Torres Pazmino et\u00a0 al. 2008). Finally, the GST tag does not appear to adversely affect the kinetic behavior of the enzyme as GSTACMO (ACMO with a fused N-terminal GST tag) elicited similar kcat and Km with cyclohexanone com-pared to ACMO. Given that chimeric protein elicits simi-lar steady state kinetic properties as ACMO and is more stable, subsequent kinetic, thermodynamic and binding studies described below were performed on GSTACMO.Molecular modellingTo structurally rationalize the low  NADP+ binding affin-ity we performed a sequence alignment of ACMO with related BVMOs and constructed a homology model of ACMO using MODELLER (version 9.20; Webb and Sali 2014). The model was created using a C65D variant of PAMO as a template (PDB entry 4d03), which shares 43% sequence identity with ACMO. The sequence alignment and ACMO model revealed sequence variation in the Table 1 Steady-state kinetic parameters of ACMO and GSTACMOThe experiments were performed in 50 mM HEPES\u2013NaOH pH 7.5 at 25 \u00b0C as described in \u201cMaterials and methods\u201d. Butanone was present at a fixed saturating concentration of 200 \u03bcM in steady-state experiments where NADPH served as the variable substrate. For experiments where the ketone served as the variable substrate, NADPH was present at 100 \u00b5MEnzyme Variable substrate kcat  (s\u22121) Km (\u00b5M) kcat\/Km  (M\u22121 s\u22121) \u00d7 103ACMO NADPH 2.0 \u00b1 0.1 6.7 \u00b1 0.8 300 \u00b1 36ACMO Acetone 1.4 \u00b1 0.2 170 \u00b1 11 8.5 \u00b1 0.59ACMO Butanone 2.1 \u00b1 0.1 0.34 \u00b1 0.03 6000 \u00b1 550ACMO 2-Pentanone 1.9 \u00b1 0.1 0.37 \u00b1 0.06 4800 \u00b1 760ACMO 2-Heptanone 3.9 \u00b1 0.1 1.5 \u00b1 0.1 2600 \u00b1 130ACMO 3-Methylbutanone 2.2 \u00b1 0.1 4.4 \u00b1 0.5 510 \u00b1 55ACMO 2,4-Dimethyl-3-pentanone 1.5 \u00b1 0.1 1500 \u00b1 220 1.0 \u00b1 0.15ACMO Cyclobutanone 2.0 \u00b1 0.1 1.5 \u00b1 0.2 1400 \u00b1 190ACMO Cyclopentanone 4.3 \u00b1 0.1 120 \u00b1 11 35 \u00b1 3.2ACMO Cyclohexanone 3.6 \u00b1 0.2 2400 \u00b1 400 1.5 \u00b1 0.3ACMO Bicyclo[3.2.0]hept-2-en-6-one 1.5 \u00b1 0.1 6.7 \u00b1 1.3 220 \u00b1 45ACMO Phenylacetone 1.0 \u00b1 0.1 8.9 \u00b1 1.5 112 \u00b1 20GSTACMO Cyclohexanone 2.8 \u00b1 0.1 2300 \u00b1 380 1.2 \u00b1 0.2H325K NADPH 1.6 \u00b1 0.1 0.48 \u00b1 0.05 3250 \u00b1 320H325K Butanone 1.6 \u00b1 0.1 0.48 \u00b1 0.05 3250 \u00b1 320Table 2 Additional kinetic parameters for ACMOThe inhibition constant for  NADP+, Ki (NADP+) was determined in the presence of 200 \u03bcM butanone. The turnover rates for uncoupled NADPH oxidation (kunc) were measured with 100 \u03bcM NADPH. The limiting rate constant for NADPH-dependent flavin reduction (kred) and the dissociation constant for NADPH (Kd (NADPH)) were determined by fitting Eq. 6 to the data shown in Fig. 4a. All reactions were performed at 25 \u00b0C in 50 mM HEPES\u2013NaOH, pH 7.5Enzyme Parameters Measured valuesACMO KI (NADP+) 166 \u00b1 13 \u00b5MH325K KI (NADP+) 27 \u00b1 6 \u00b5MACMO kunc  (s\u22121) 0.26 \u00b1 0.02 s\u22121ACMO kred  (s\u22121) 59 \u00b1 3 s\u22121ACMO Kd (NADPH) 120 \u00b1 14 \u03bcMPage 7 of 13Fordwour et al. AMB Expr           (2018) 8:181 coenzyme binding cleft (Fig.\u00a02). Typically, a lysine residue interacts with the 2\u2032-phosphate of NADP(H) in BVMOs. This noncovalent interaction has been shown to improve the binding affinity for the coenzyme in addition to the enzyme\u2019s preference for NADPH over NADH (Kamer-beek et\u00a0 al. 2004). In ACMO, a histidine residue  (His325) replaces the lysine, a substitution that could potentially weaken the electrostatic interaction between the 2\u2032 phosphate of the coenzyme and the protein. To test this hypothesis,  His325 was substituted for a lysine. As shown in Tables\u00a01 and 2, the H325K substitution lead to a signif-icant improvement in coenzyme binding affinity as evi-denced by the sixfold decrease in the Km for NADPH and a 14-fold decrease in Ki for  NADP+. The modeling exer-cise also revealed the insertion of an alanine (A433) in an active site bulge that has been shown to restrict the sub-strate scope of PAMO (Bocola et\u00a0al. 2005; Reetz and Wu 2008). Deletion of this residue in ACMO led to a 130-fold decrease in the kcat\/Km for butanone, whilst the catalytic efficiency for cyclobutanone was unchanged. There was no increase in NADPH oxidation in the presence of ace-tone for this variant.NADP+ binding affinity for GSTACMOThe binding of  NADP+ to PAMO and CHMO has been shown to induce notable shifts in the flavin absorbance spectra. This absorbance shift is attributed to displace-ment of a highly conserved catalytic arginine residue by the  NADP+ nicotinamide ring as it docks over the xylene portion of the FAD isoalloxazine ring (Torres Pazmino et\u00a0al. 2008; Sheng et\u00a0al. 2001). In wild type CHMO and PAMO, binding of  NADP+ causes the absorbance peak at 383 to shift to 366\u00a0nm and the peak at 440\u00a0nm to develop a more prominent absorbance shoulder at 480\u00a0 nm. In GSTACMO, there is also a blue shift in the absorb-ance peak at 380\u00a0 nm similar to that of related BVMOs (Fig.\u00a0 3). However, in GSTACMO, the peak at 440\u00a0 nm shifts to 450\u00a0nm with a sizable shoulder at 430\u00a0nm. The distinct spectral shifts observed in GSTACMO are likely to due\u00a0 to minor structural variations in the active site induced by the binding of the oxidized coenzyme. To determine the binding affinity of the coenzyme, the absorbance change at 467\u00a0 nm was plotted against the concentration of  NADP+ and the data were fitted to the equation describing the quadratic binding isotherm. For wild type GSTACMO, the dissociation constant for the GSTACMO-NADP+ complex was 21.1 \u00b1 4.1\u00a0 \u03bcM where as for the H325K variant it was 0.50 \u00b1 0.04\u00a0\u03bcM.Reduction potential of the ACMO flavin cofactorThe reduction potential of the enzyme was determined by reducing GSTACMO in the presence of indigo disul-fonate, which served as a reference dye. Figure\u00a04a shows the combined spectra of the dye and GSTACMO fol-lowing incubation with the xanithine\/xanthine oxidase reducing system and the redox mediator benzyl viologen. The concentrations of the oxidized and reduced forms of the dye and GSTACMO were determined at 610 and 458\u00a0nm, respectively, as described under\u00a0\u201cMaterials and Methods\u201d. A plot of log (Ered\/Eox) versus log (Dred\/Dox) has a slope of 1.0, which indicates that an equal number of electrons (i.e. two) were transferred between GSTACMO and indigo disulfonate (Fig.\u00a0 4b). The midpoint potential of GSTACMO calculated from the y-intercept of the HAPMO   433 PDS-PVGGKRIVRDCHMO    320 PQDL--YAKRPLCD      427  GPNGP---FTNL436  GPGSPS-ALSNM PAMO    328 PKGYPFGTKRLILE   ACMO    317 PSDYGFGTHRVPLE 426 VPLAPSAALCNM His325 Ala433Ala434Ser432Lys336Ala442Ser441a b c Fig. 2 Comparison of the crystal structure of PAMO (a, PDB entry 2YLR) and a homology model of ACMO (b) depicting distinct residues neighboring the 2\u2032-phosphate of  NADP+ and the FAD and  NADP+ are shown as stick models with the carbon atoms in yellow and cyan, respectively. c A sequence alignment of BVMOs showing variation in the active sites. Sequence variation in ACMO is highlighted in blue and redPage 8 of 13Fordwour et al. AMB Expr           (2018) 8:181 a b cd Fig. 3 Flavin absorption spectra of GSTACMO (a) and H325K (b) were recorded in 50 mM HEPES\u2013NaOH pH 7.5 upon titration with  NADP+ (0\u2013250 \u03bcM). The inset shows the difference spectra. Absorbance changes at 467 nm for wild type GSTACMO (c) and H325K (d) were plotted as a function of  NADP+ concentration and the data were fitted to a quadratic binding isotherm (Eq. 1), which gave Kd values of 21.1 \u00b1 4.1 \u03bcM (wild type) and 0.50 \u00b1 0.04 \u03bcM (H325K)a bFig. 4 Reduction potential measurement of GSTACMO. a The combined absorbance spectra of GSTACMO and indigo disulfonate as both species are slowly reduced with xanthine\/xanthine oxidase. The standard reduction potential value of the enzyme (E\u00b0), calculated from the y-intercept of the plot of log (Ered\/Eox) versus log (Dred\/Dox) shown in b, is \u2212 166 mV \u00b1 1 mVPage 9 of 13Fordwour et al. AMB Expr           (2018) 8:181 graph is \u2212\u00a0 166 \u00b1 1\u00a0 mV. Thus, GSTACMO is thermody-namically poised to accept a hydride ion from NADPH, which has a midpoint potential of \u2212\u00a0320\u00a0mV.pH\u2011dependence and thermostabilityThe thermostability of GSTACMO was measured by pre-incubating the enzyme at various temperatures (5\u201345\u00a0\u00b0C) for one hour and then measuring the residual activity at 25\u00a0\u00b0C (Fig.\u00a05a). The enzyme elicited comparable residual activity at temperatures \u2264 20\u00a0 \u00b0C. However, incubation of the enzyme at temperatures 25\u00a0\u00b0C and above resulted in > 60% loss of enzyme activity. These results demon-strate that GSTACMO is a relatively unstable protein. The pH-dependence profiles show a bell-shaped curve with maximal activity between pH 7 and 8 (Fig.\u00a05b). The pKa values of the ionizable groups responsible for optimal activity were determined by fitting the data to Eq.\u00a05. The fitting routine revealed that enzyme activity is dependent on protonation of a single ionizable group with of pKa of 5.6 \u00b1 0.2 and ionization of group with a pKa of 9.2 \u00b1 0.3.Reductive\u2011half reactionThe rate of NADPH-dependent reduction of GSTACMO was measured by following the absorbance decrease at 443\u00a0nm upon rapid mixing\u00a0of the enzyme with a tenfold excess of coenzyme (Fig.\u00a06a). The monophasic decay was fitted to a standard single exponential equation, which gave an observed rate constant (kobs) of 28.4 \u00b1 0.5\u00a0 s\u22121. To confirm that this initial kinetic phase involved trans-fer of a hydride ion from NADPH to the FAD cofactor, the reaction was repeated with 4(R)-[4-2H]NADPH. As shown in Fig.\u00a06a, reduction of the enzyme with the deu-terated coenzyme was significantly slower and a fit of the a bFig. 5 a Residual activity of GSTACMO following a 1 h pre-incubation of the enzyme at various temperatures. b pH-dependence of GSTACMO activity. Reactions were performed as described in \u201cMaterials and methods\u201da b Fig. 6 Single-wavelength stopped-flow experiments. a Stopped-flow single wavelength absorbance (443 nm) after mixing 20 \u03bcM of GSTACMO with 200 \u03bcM of NADPH or 4(R)-[4-2H]NADPH. Absorbance traces (black lines) were collected over 1 s (a) and fitted to standard single exponential equation (grey dotted line), which gave values of kred1 of 28.4 \u00b1 0.5 s\u22121 (NADPH) and 7.5 \u00b1 0.2 s\u22121 (4(R)-[4-2H]NADPH). b Dependence of kred1 on the concentration of NADPH of wild type ACMO (solid circles) and the H325K variant (open circles). The data were fitted to Eq. 6 and the results are shown in Table 2Page 10 of 13Fordwour et al. AMB Expr           (2018) 8:181 absorbance trace to a single exponential equation pro-duced an observed rate constant of 7.5 \u00b1 0.2\u00a0 s\u22121 and a kinetic isotope effect of 3.8 \u00b1 0.2.Single-wavelength stopped-flow experiments were also used to determine if kobs was affected by NADPH con-centration. The stopped-flow absorbance changes were followed at 443\u00a0nm over 0.5\u00a0s and were fitted to a single exponential equation at concentrations of NADPH that were at least sevenfold greater than that of the enzyme concentration. The observed rate constant, kobs, exhib-ited a hyperbolic saturation dependence on NADPH concentration, and a fit of the data to Eq.\u00a06 yielded a Kd of 121 \u00b1 14\u00a0\u03bcM and a kred (maximal rate constant of fla-vin reduction) of 59 \u00b1 3\u00a0s\u22121 (Table\u00a02; Fig.\u00a06b). The H325K elicited a similar rate of NADPH-dependent flavin reduc-tion as the wild type enzyme; however, the observed rate constant was not dependent on NADPH concentration, which is consistent with this variant having a higher affinity for the coenzyme.Oxidative\u2011half reactionStopped-flow spectroscopy was used to monitor the catalytic events of the oxidative half-reaction. In the first experiment, the reduced enzyme in the presence of 500\u00a0 \u03bcM  NADP+ was mixed with air-saturated buffer in the absence of ketone substrate. These mixing conditions lead to the formation of the C4a-peroxyflavin interme-diate, which would subsequently decay to  H2O2 and the fully oxidized flavin cofactor. For GSTACMO, the absorb-ance spectra were collected over 1.5 and 75\u00a0 s on a log-time base and then subsequently combined; a selection of these spectra are shown in Fig.\u00a07a. The time resolved spectra show initial formation of an absorbance peak at 366\u00a0 nm, demarking formation of the C4a-peroxyflavin intermediate following  O2 activation by the reduced FAD cofactor. The absorbance at 440\u00a0 nm subsequently increases reflecting decomposition of the C4a-perox-yflavin intermediate and reformation of the oxidized FAD cofactor. If the reaction is repeated with the pres-ence of 200\u00a0\u03bcM butanone in the air-saturated buffer, then formation of the C4a-peroxyflavin intermediate is less obvious, likely owing to its rapid decomposition in the presence of the carbonylic substrate (Fig.\u00a07b). To extract rate constants for the kinetic events associated with the oxidative half reaction, we switched to single-wavelength mode with the stopped-flow apparatus, which enabled us to average multiple traces and acquire earlier data time points. In the absence of butanone, the absorbance changes at 366\u00a0nm were fit to a standard single exponen-tial equation which gave an observed rate constant of 8.9 \u00b1 0.2\u00a0s\u22121, while the observed rate constants at 440\u00a0nm were 0.47 \u00b1 0.02\u00a0 s\u22121 (kox1) and 0.06 \u00b1 0.01\u00a0 s\u22121 (kox2; Table\u00a03). When the reaction was repeated in the presence of 200\u00a0\u03bcM butanone, then the observed rate constant at 366\u00a0nm was 6.5 \u00b1 0.8\u00a0s\u22121 and at 440\u00a0nm, the single kinetic phase gave an observed rate constant 0.80 \u00b1 0.01\u00a0 s\u22121 (k440). The observed rate constant for the formation of C4a-peroxyflavin at 366\u00a0nm was shown to increase with oxygen concentration (Fig.\u00a0 8). From a linear fit of the observed rate constant versus oxygen concentration, we obtained a second order rate constant of 49\u00a0 mM\u22121\u00a0 s\u22121 (koo-) for the reaction between  O2 and the reduced flavin. A similar bimolecular rate constant (koo-BT) was obtained in the presence of butanone.  DiscussionGordonia sp. strain TY-5 is a bacterium that is capable of utilizing gaseous propane as the sole course of carbon during aerobic growth. ACMO is part of the propane degradation pathway present in the bacterium, for which acetone is a central intermediate. Although ACMO was initially identified as acting in the catabolism of acetone (Kotani et\u00a0 al. 2007), the enzyme elicits a 700-fold and 165-fold lower catalytic efficiency towards this substrate relative to butanone and cyclobutanone respectively. Although PAMO shares 43% sequence similarity with ACMO, PAMO does not consume acetone (de Gonzalo et\u00a0al. 2005; Fraaije et\u00a0al. 2005). Instead, PAMO preferen-tially oxidizes small aromatic ketones, such as phenylac-etone. Mutagenesis studies of PAMO point to an active site loop comprising residues 440\u2013446 as a structural feature that influences substrate specificity (Bocola et\u00a0al. 2005; Reetz and Wu 2008). Deletion of residues S441 and A442 (PAMO numbering) resulted in an enzyme variant that was able to accept bulkier substrates (Bocola et\u00a0 al. 2005). Sequence alignment of PAMO and ACMO reveals that insertion of an alanine residue between S441 and A442, which may be a structural adaptation that restricts the ACMO active site and increase its catalytic efficiency towards smaller ketones such as acetone (Fig.\u00a0 2). In ACMO, this residue (A433) improved catalytic efficiency towards small aliphatic ketones.Stopped-flow spectroscopy was used to measure the rates of the reductive and oxidative half reactions. NADPH-dependent reduction of the ACMO FAD cofac-tor was relatively fast with an observed rate constant of ~ 60\u00a0s\u22121 at 25\u00a0\u00b0C. This rate constant is ~ 3\u20135-fold faster than that observed for CHMO and PAMO under simi-lar experimental conditions (Ryerson et\u00a0 al. 1982; Torres Pazmino et\u00a0 al. 2008). However, the oxidative half reac-tion, particularly the formation of the C4a-peroxyfla-vin intermediate was considerably slower in ACMO. In CHMO and PAMO, the C4a-peroxyflavin adduct forms in < 10\u00a0 ms at final  O2 concentration of ~ 0.3\u00a0 mM, and the second-order rate constant was determined to be 870\u00a0mM\u22121\u00a0s\u22121 for PAMO and 1700\u00a0mM\u22121  s\u22121 for Page 11 of 13Fordwour et al. AMB Expr           (2018) 8:181 CHMO. In contrast, the second order rate constant for the reaction between  O2 and the reduced FAD in ACMO (49\u00a0 mM\u22121  s\u22121) was 20\u201330-fold lower. The C4a-perox-yflavin is also not as stable in ACMO even in the pres-ence of saturating amounts of  NADP+ as it decayed with a rate constant of 0.47\u00a0 s\u22121 in the absence of substrate. The instability of the C4a-peroxyflavin intermediate likely accounts for the relatively high rate of uncoupled NADPH oxidation (0.26\u00a0s\u22121) in ACMO. The NADPH oxi-dase activity of CHMO and PAMO is 50-fold and 150-fold lower, respectively, than that of the monooxygenase activity (Torres Pazmino et\u00a0 al. 2008; Sheng et\u00a0 al. 2001), but in ACMO it is only tenfold lower. It is unclear why a b d c Fig. 7 a Time-resolved absorbance spectra collected over 75 s following the re-oxidation of reduced GSTACMO (20 \u03bcM) in the presence 500 \u03bcM  NADP+ with air-saturated buffer (50 mM HEPES pH 7.5, 20% glycerol). b The same experiment as performed as for panel A with the exception of 200 \u03bcM of butanone added to the air-saturated buffer. c Stopped-flow single wavelength traces at 366 nm following rapid mixing of reduced GSTACMO (20 \u03bcM) in the presence 500 \u03bcM  NADP+ against air-saturated buffer (50 mM HEPES pH 7.5, 20% glycerol) with 200 \u03bcM of butanone (black circles) and without butanone (grey circles). Fitting the data to a single exponential equation gave observed rate constants of 6.5 \u00b1 0.8 s\u22121 (with butanone) and 8.9 \u00b1 0.2 s\u22121 (without butanone). d Stopped-flow single wavelength traces at 440 nm following rapid mixing of reduced GSTACMO (20 \u03bcM) in the presence 500 \u03bcM  NADP+ against air-saturated 50 mM HEPES pH 7.5, 20% glycerol with 200 \u03bcM of butanone (black circles) and without butanone (grey circles). The absorbance traces with butanone were fitted to a single exponential equation giving an observed rate constant of 0.80 \u00b1 0.01 s\u22121, and the absorbance traces without butanone were fitted to a double exponential giving rate constants of 0.47 \u00b1 0.01 s\u22121 and 0.06 \u00b1 0.01 s\u22121Table 3 Observed rate constants for the oxidative half reaction. The reactions were performed in 50 mM HEPES\u2013NaOH, pH 7.5 at 25 \u00b0Ca Determined from the slopes of a plot of the observe rate constants for the absorbance changes at 366 nm versus the concentration of dioxygen (Fig. 8)b Determined from fitting a double exponential to the grey single wavelength absorbance trace at 440 nm shown in Fig. 7c Determined from fitting a single exponential to the black single wavelength absorbance trace at 440 nm shown in Fig. 7Without butanone With butanonek\u2212OO  (mM\u22121  s\u22121)akox1  (s\u22121)b kox2  (s\u22121)b kOO\u2011BT  (mM\u22121  s\u22121)ak440  (s\u22121)c40.1 \u00b1 5.1 0.47 \u00b1 0.01 0.06 \u00b1 0.01 s\u22121 44.2 \u00b1 9.2 0.80 \u00b1 0.01Page 12 of 13Fordwour et al. AMB Expr           (2018) 8:181 this may be the case in ACMO, but it may be linked to the enzyme\u2019s relatively weak binding affinity for  NADP+.Steady-state inhibition studies revealed that the  NADP+ binding affinity is ~ 166\u00a0 \u03bcM, similar to the dis-sociation constant for NADPH (120\u00a0 \u03bcM) determined through stopped-flow experiments. By way of contrast, the Kd for NADPH is 0.7\u00a0\u03bcM and 11\u00a0\u03bcM for PAMO and CHMO, respectively (Torres Pazmino et\u00a0 al. 2008; Ryer-son et\u00a0 al. 1982). Interestingly, the dissociation constant for the oxidized coenzyme determined through titration experiments was significantly lower at 21\u00a0\u03bcM. It unclear why a discrepancy is observed in the experimentally determined Kd values. It may have to do with the oxida-tion state of the coenzyme or enzyme. The stopped flow experiments measure the binding affinity of NADPH for the oxidized enzyme, steady-state inhibition studies measure the binding affinity of  NADP+ for the reduced enzyme and the titration experiments measure the bind-ing affinity of  NADP+ to the oxidized enzyme. Previous studies have experimentally shown that the redox state of the enzyme can modulate the binding affinity for the pyridine nucleotide (van den Heuvel et\u00a0al. 2005).Crystal structures of CHMO and PAMO in com-plex with  NADP+ show the coenzyme in an extended conformation, wedged between the Rossmann-fold of the NADP-domain and a loop within the FAD domain (Fig.\u00a02). Residues coordinated to the coenzyme in PAMO are conserved in ACMO, with the exception of  Lys336 (PAMO numbering), which coordinates to the 2\u2032-phos-phate of the coenzyme. Mutagenesis of the lysine in HAPMO  (Lys429) demonstrates its importance in increas-ing the binding affinity of NADPH and ensuring that the enzyme preferentially selects for NADPH over NADH. In ACMO, the lysine to histidine substitution does not affect the enzyme\u2019s preference for NADPH over NADH as both the wild type enzyme and the H325K variant were unable to catalyze the oxidation of NADH. The sub-stitution does however strengthen the enzyme\u2019s affinity for the 2\u2032-phosphorylated coenzyme by sixfold.In summary, we have shown that AMCO elicits the highest catalytic efficiency towards butanone and cyclob-utanone. The enzyme also elicits a relatively weak bind-ing affinity for NADP(H), which is partially attributed to sequence variation in the 2\u2032-phosphate binding pocket. Reduced ACMO also reacts more slowly with  O2 and is less efficient at stabilizing the C4a-peroxyflavin adduct compared to related BVMOs. As a consequence, ACMO elicits a relatively high NADPH oxidase activity com-pared to its monooxygenase activity. Finally, kinetic iso-tope studies support transfer of the proR hydrogen. This implies that the re-face of the nicotinamide ring is plan-ner with the FAD for reductive half reaction. Following hydride transfer the enzyme and cofactor undergo a con-formational switch that places the si-face of the nicotina-mide ring over the FAD, in position to stabilize reactive oxygenating intermediates of the oxidative half reaction.Additional fileAdditional file 1: Fig.S1. A 10 % SDS-PAGE gel of recombinant ACMO. Lane 1, molecular weight markers; lane 2, crude extract; lane 3, glutathione-sepharose showing the GST-ACMO chimera and lane 4, recombinant ACMO following Q-sepharose chromatography.Authors\u2019 contributionsOBF performed all the experiments and data analysis, KRW designed the experiments and wrote the manuscript, GL purified protein and MH initiated the project and provided supervision. All authors read and approved the final manuscript.Author details1 Department of Chemistry, University at the British Columbia, Okanagan Campus, 3247 University Way, Kelowna, BC V1V 1V7, Canada. 2 School of Engi-neering, University at the British Columbia, Okanagan Campus, 3247 University Way, Kelowna, BC V1V 1V7, Canada. Competing interestsThe authors declare that they have no competing interests.Availability of data and materialsAll relevant data are presented in the main paper.Consent for publicationNot applicable.Ethics approval and consent to participateNot applicable.FundingThis work is supported by a grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2015-06130).Fig. 8 The dependence of the kobs1 measured at 366 nm on  [O2]. The observed rate constants were plotted as a function of  [O2] in the absence (closed squares) and presence of 200 \u03bcM butanone (closed circles) for GSTACMO. The slope of the lines were used to determine the bimolecular rate constants (koo- and koo-BT) shown in Table 3Page 13 of 13Fordwour et al. AMB Expr           (2018) 8:181 Publisher\u2019s NoteSpringer Nature remains neutral with regard to jurisdictional claims in pub-lished maps and institutional affiliations.Received: 23 August 2018   Accepted: 29 October 2018ReferencesBalke K, Kadow M, Mallin H, Sass S, Bornscheuer UT (2012) Discovery, applica-tion and protein engineering of Baeyer\u2013Villiger monooxygenases for organic synthesis. Org Biomol Chem 10(31):6249\u20136265. https :\/\/doi.org\/10.1039\/c2ob2 5704a Bocola M, Schulz F, Leca F, Vogel A, Fraaije MW, Reetz MT (2005) Converting phenylacetone monooxygenase into phenylcyclohexanone monooxy-genase by rational design: towards practical Baeyer\u2013Villiger monooxyge-nases. Adv Synth Catal 347(7\u20138):979\u2013986Bowman MJ, Jordan DB, Vermillion KE, Braker JD, Moon J, Liu ZL (2010) Stereo-chemistry of furfural reduction by a Saccharomyces cerevisiae aldehyde reductase that contributes to in situ furfural detoxification. 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