| The α/β-hydrolase fold family of enzymes is rapidly becoming one of the largest groups of structurally related enzymes with diverse catalytic functions. The enzymes all have a Nucleophile-His-Acid catalytic triad evolved to efficiently operate on substrates with different chemical composition or physicochemical properties and in various biological contexts. As one of the superfamily, HOPDA hydrolases (2-Hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic Acid hydrolases, BphD) is a C-C bond cleavage hydrolase which hydrolyzes a ring-cleavage product of an aromatic compound generated in a biphenyl/polychlorinated biphenyl (PCB) degradation pathway of bacteria. In recent years, the study of its catalytic mechanism has become the hot research field.According to reports and preliminary researches in our laboratory, six mutants were constructed to investigate the catalytic mechanism of the C-C hydrolase. The expression products were purified by Q Sepharose (Hitrap Q) and Sephacryl S-300. Replacements of Ser-110, Asp-237and His-265in HOPDA hydrolase with Ala led to17～19-fold decreases in kcat, and showed104～103-flod reduced kcat/Km values compared with those of the wild-type enzyme; but, the same replacements for Trp-85and Trp-219of HOPDA hydrolase led to3.6-and3.3-fold decreases in kcat/Km, respectively. However, as a mutant of tryptophan, mutation of Trp-266to Ala yielded104-fold reduced kcat/Km. Circular dichroism of S110A, D237A, H265A and W266A show that a-helix is dominant, and consistent with that of the wild-type enzyme. The results of chemical modification shows that the chemical modifiers had no influence on the mutants S110A, D237A, H265A and Trp266, but0.5mmol/L of NBS reduced the activity of W85A by90%and W219A by70%. The results indicate that Ser110, Asp237and His265are necessary for the catalytic reaction of HOPDA hydrolase, and Trp266plays a key role in the enzymatic reaction. Pre-steady-state kinetic analysis of mutant S110A, H265A and W266A were determined, and the enzyme-substrate complex (E:S) of mutant S110A and H265A were identified through the scanning spectrometry, either. Three steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation, C-C cleavage and product release: S110A exhibit fast ketonisation (k1492=100s-1), an intermediate phase, slow C-C cleavage and product release (k2492=0.32s-1, k3492=0.027s--1); The Stop-flow data of mutant W266A was similar to S110A (k1492=45.45s-1, k2492=0.27s-1, k3492=0.15s--1); H263A exhibits very slow ketonisation and C-C cleavage(k1492=0.02s-1, k2492=019s-1). The above results showed, His265functions as contributors to catalysis of the initial enol/keto tautomerisation of the substrate and C-C cleavage; mutant Ser110is responsible for C-C cleavage, which also was proved by the intermediate (E:S) of the muants. Similarly, the stop-flow data of mutant W266A implicated that, in Rhodococcus sp. R04BphD, Trp-266is important to the C-C cleavage and the product release.Random mutagenesis on Rhodococcus sp. R04BphD gene was conducted by using error-prone PCR strategy. The sequence comparison showed that there were two bases, L61, P253, had been replaced in the most of sequences, and the mutation was fuound mostly in the rage0-40,60-100and160-210in the structure-based sequence of BphD. In order to improve the screening efficiency, we established a fast and efficient screen method called "96-well plates screen method". It is hopeful that we can obtain a new enzyme with wider scope of substrate or higher activity through the experiment, which could provided a basis to further improve of the BphD catalytic efficiency. |
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