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Mechanistic and proteomic studies of carbohydrate-active enzymes : β-1,4-glycanases and colletotrichum lindemuthianum chitin deacetylase Hekmat, Omid

Abstract

The field of proteomics is facing many challenges, one of which is the assignment of structure and function to all of the proteins encoded and expressed by prokaryotic and eukaryotic genomes. One approach to this involves the development of new methodologies for the activity-based protein profiling (ABPP) of enzymes. Glycoside hydrolases (GH) are one group of enzymes that still lack an efficient and specific mass spectrometry-based ABPP (MS-ABPP) method. A probe was synthesized which consists of a biotin tag, a disulfide-bonded spacer, and a 2-deoxy-2-fluoro-xylobioside inactivator that covalently labels retaining xylanases and mixed-function cellulases. The MS-ABPP methodology was demonstrated at three levels of increasing complexity. Firstly, the labelled active-site peptides of a Bacillus circulans endo-xylanase (Bex) and a Cellulomonas fimi mixed-function endo-xylanase/cellulase (Cex) were affinity-isolated from their proteolytic digests and sequenced by ESI-MS/MS, leading to the identification of the enzymes' catalytic nucleophiles, Glu₇₈ (Bex) and Glu₂₃₃ (Cex). Secondly, the method was applied to several model enzyme mixtures including either Bex or Cex or both as targets. Finally, the method was used to analyze the secreted proteome of the soil bacterium, Cellulomonas fimi. The labelled active-site peptide (VQITEL) of a new GH family 10 glycanase was affinity-isolated from the proteolytic digest of the proteome and sequenced by ESI-MS/MS and Edman degradation. The glycanase gene was cloned using inverse PCR techniques and the protein was found to comprise a catalytic domain that shares about 70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. Carbohydrate O-esterases and N-deacylases (CE) are a class of carbohydrateactive enzymes whose catalytic mechanisms have not been vigorously investigated. A detailed mechanistic investigation was conducted on Colletotrichum lindemuthianum endo-type chitin deacetylase (CDA) from CE family 4. Steady-state kinetic and mass spectrometric analyses showed that CDA has four sugar-binding subsites (-2, -1, 0, and +1). Subsites -2 and +1 make major contributions to the overall substrate-binding free energy change (A₋₂ = -11 and A₊₁ = -13 kJ.mol⁻¹) and subsites -2 and 0 (reaction site) recognize the acetamido group (ΔΔG[sub (N-acetyl)] = 3 and 4 kJ.mol⁻¹, respectively). Hammett linear free energy correlations using α-haloacetamido substrate analogues demonstrated the presence of an oxyanion intermediate (positive slope) and significant negative charge development in the transition state (ρ = 2). Since this oxyanion intermediate cannot have an enolate structure (an elimination/addition reaction was rejected), the only other possibility is that of a tetrahedral structure in a substitution reaction. A classical bellshaped pH dependence of k[sub cat]/K[sub m] (pK[sub al] = 7.5, pK[sub a]₂ = 10) revealed an active site environment far different from those of carboxyl proteases (both pK[sub a] values in the acidic pH range). Kinetic and ICP-MS results indicated the lack of a structural/catalytic divalent metal cofactor. The lack of covalent inactivation of CDA by a carbamate ester and by α -haloacetamido substrate analogues is consistent with direct water attack in a single-step substitution but these results do not conclusively rule out the possibility of a double-step substitution. Structural information is required to elucidate the exact role of the conserved residues in the active site of CDA.

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