Khomaini Hasan

Biochemical Characterization of Haloalkane Dehalogenases DrbA and DmbC, Representatives of a Novel Subfamily

ABSTRACT This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.

Biochemical Characteristics of the Novel Haloalkane Dehalogenase DatA, Isolated from the Plant Pathogen Agrobacterium tumefaciens C58

ABSTRACT We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.

The effect of a unique halide-stabilizing residue on the catalytic properties of haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

Haloalkane dehalogenases catalyze the hydrolysis of carbon–halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide‐stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan–asparagine pair. The newly‐isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide‐stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre‐steady‐state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen‐bonding patterns within the active sites of the wild‐type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7‐fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K0.5 for selected halogenated substrates by a factor of 2–4, without impacting the rate‐determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.

Chromatography as the Major Tool in the Identification and the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64

© 2012 Ismaya et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chromatography as the Major Tool in the Identification and the Structure-Function Relationship Study of Amylolytic Enzymes from Saccharomycopsis Fibuligera R64

Computational Model of the Effect of a Surface-Binding Site on the Saccharomycopsis fibuligera R64 α-Amylase to the Substrate Adsorption

α-Amylase is one of the important enzymes in the starch-processing industry. However, starch processing requires high temperature, thus resulting in high cost. The high adsorptivity of α-amylase to the substrate allows this enzyme to digest the starch at a lower temperature. α-Amylase from Saccharomycopsis fibuligera R64 (Sfamy R64), a locally sourced enzyme from Indonesia, has a high amylolytic activity but low starch adsorptivity. The objective of this study was to design a computational model of Sfamy R64 with increased starch adsorptivity using bioinformatics method. The model structure of Sfamy R64 was compared with the positive control, ie, Aspergillus niger α-amylase. The structural comparison showed that Sfamy R64 lacks the surface-binding site (SBS). An SBS was introduced to the structure of Sfamy R64 by S383Y/S386W mutations. The dynamics and binding affinity of the SBS of mutant to the substrate were also improved and comparable with that of the positive control.