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Biochemical and crystallographic studies of unusual imino-acid-reducing enzymes Guo, Jin
Abstract
Chiral amine moieties are widely distributed in bioactive natural products and pharmaceutical ingredients. NAD(P)H-dependent imine reductases (IREDs) have been identified as potential biocatalysts for chiral amine synthesis via asymmetric reduction of the imine substrates. In this work, I characterized two unusual imino-acid-reducing enzymes, Punc5 and Bsp5, from the D-2-hydroxy-acid dehydrogenase (DHDH) family. The DHDH enzymes are known for reducing α-keto acids directly to the corresponding chiral hydroxy acids; however, both Punc5 and Bsp5 demonstrate imine reductase activity. Specifically, when coupled with L-arginine oxidase Ind4, both enzymes can use the coenzyme NAD(P)H to stereo-specifically reduce the Ind4 products didedydroarginine and dedydroarginine to D-4,5-dehydroarginine and D-arginine, respectively. Furthermore, Punc5 shows a DHDH activity, converting 2-ketoarginine to 5-guanidino-2-hydroxypentanoic acid. Both IREDs and DHDHs belong to the NAD(P)H-dependent oxidoreductase family; however, imine reduction catalyzed by DHDHs had never been reported before. To understand how Punc5 and Bsp5 evolved from DHDHs with asymmetric imino-acid-reducing activities, and to offer insights into NAD(P)H-dependent oxidoreductases’ chemoselectivity, I obtained ~1.6 Å resolution ternary structures of each enzyme bound with coenzyme NADPH and product D-arginine. These ternary structures of Punc5 and Bsp5 at high resolution closely resemble typical DHDHs; however, the spatial relationship of the coenzyme, product, and catalytic residues within the active site suggests a different catalytic mechanism from typical DHDHs. Structure-guided mutagenesis work uncovered an essential residue Tyr97 for substrate binding in Punc5. Biochemical characterization of the Punc5-Y97F variant suggests imine reduction under the acidic condition is a more facile reaction compared to ketone reduction as Punc5-Y97F is active towards imino acids, but it is inactive towards 2-ketoarginine. This unique imino-acid-reducing activity demonstrated by Punc5 and Bsp5 indicate that other subfamilies of NAD(P)H-dependent oxidoreductases besides known IREDs could also have the potential to produce chiral amines and be applied in pharmaceutical industry. Besides, our work offered three-dimensional frameworks for understanding how these unusual imino acid reductases differ from typical DHDHs, setting the stage for further engineering efforts to either enhance their catalytic efficiency or expand their substrate scopes.
Item Metadata
Title |
Biochemical and crystallographic studies of unusual imino-acid-reducing enzymes
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
2018
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Description |
Chiral amine moieties are widely distributed in bioactive natural products and pharmaceutical ingredients. NAD(P)H-dependent imine reductases (IREDs) have been identified as potential biocatalysts for chiral amine synthesis via asymmetric reduction of the imine substrates. In this work, I characterized two unusual imino-acid-reducing enzymes, Punc5 and Bsp5, from the D-2-hydroxy-acid dehydrogenase (DHDH) family. The DHDH enzymes are known for reducing α-keto acids directly to the corresponding chiral hydroxy acids; however, both Punc5 and Bsp5 demonstrate imine reductase activity. Specifically, when coupled with L-arginine oxidase Ind4, both enzymes can use the coenzyme NAD(P)H to stereo-specifically reduce the Ind4 products didedydroarginine and dedydroarginine to D-4,5-dehydroarginine and D-arginine, respectively. Furthermore, Punc5 shows a DHDH activity, converting 2-ketoarginine to 5-guanidino-2-hydroxypentanoic acid. Both IREDs and DHDHs belong to the NAD(P)H-dependent oxidoreductase family; however, imine reduction catalyzed by DHDHs had never been reported before. To understand how Punc5 and Bsp5 evolved from DHDHs with asymmetric imino-acid-reducing activities, and to offer insights into NAD(P)H-dependent oxidoreductases’ chemoselectivity, I obtained ~1.6 Å resolution ternary structures of each enzyme bound with coenzyme NADPH and product D-arginine. These ternary structures of Punc5 and Bsp5 at high resolution closely resemble typical DHDHs; however, the spatial relationship of the coenzyme, product, and catalytic residues within the active site suggests a different catalytic mechanism from typical DHDHs. Structure-guided mutagenesis work uncovered an essential residue Tyr97 for substrate binding in Punc5. Biochemical characterization of the Punc5-Y97F variant suggests imine reduction under the acidic condition is a more facile reaction compared to ketone reduction as Punc5-Y97F is active towards imino acids, but it is inactive towards 2-ketoarginine.
This unique imino-acid-reducing activity demonstrated by Punc5 and Bsp5 indicate that other subfamilies of NAD(P)H-dependent oxidoreductases besides known IREDs could also have the potential to produce chiral amines and be applied in pharmaceutical industry. Besides, our work offered three-dimensional frameworks for understanding how these unusual imino acid reductases differ from typical DHDHs, setting the stage for further engineering efforts to either enhance their catalytic efficiency or expand their substrate scopes.
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Genre | |
Type | |
Language |
eng
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Date Available |
2020-05-31
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Provider |
Vancouver : University of British Columbia Library
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Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
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DOI |
10.14288/1.0366202
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
2018-09
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Campus | |
Scholarly Level |
Graduate
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Rights URI | |
Aggregated Source Repository |
DSpace
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Rights
Attribution-NonCommercial-NoDerivatives 4.0 International