The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase isolated from Bacillus laterosporus J1 is capable of hydrolyzing GL-7-ACA and GL-7-ADCA to glutaric acid and the corresponding beta-lactam rings. Traditionally, J1 acylase was classified as class V of GL-7-ACA acylase (GCA). However, the amino acid sequence of J1 acylase has lower than 5% homology to acylases isolated from Pseudomonas strains. J1 acylase consists of a single peptide of molecular weight ∼78 kDa, in contrast with the heterodimeric nature of other cephalosporin acylases. Previous studies on this enzyme described only the specific activity, substrate preference, pH optimum and thermostability. Its tertiary structure and catalytic mechanism were not investigated in detail. It is interesting that the J1 acylase showed totally different structure from other classes of acylases but possessed the same hydrolytic activity towards cephalosporins. Homolog search revealed that J1 acylase showed 25% to 35% sequence identity to several alpha/beta-hydrolases including cocaine esterase (CocE) and alpha-amino acid ester hydrolases (AEHs). The putative catalytic triad residues conserved in J1 acylase were S125, D264 and H309, while the oxyanion-hole residues were Y57, Y126 and W173. The putative catalytic S125 was located within a highly conserved motif GXS&barbelow;YXG observed among S-15 peptidases. Secondary structure analysis had revealed alpha/beta-hydrolase fold at the N-terminal region. The catalytically important residues were located at positions where corresponding residues were found in alpha/beta-hydrolases. Tertiary structure was elaborated by homology modeling based on the X-ray structures of CocE and Acetobacter turbidans AEH (Pdb entries: 1JU3 and 1NX9, respectively). The models had demonstrated the three structural domains observed in CocE, with the putative catalytic triad residues positioned at the bottom of the active site cleft. Other catalytically important residues were identified according to the amino acid sequence alignments and residue superimpositions in tertiary structural model. Parallel site-directed mutagenesis experiments on these sites were performed to validate their functions. The mutants S125A, D264A, H309A, Y57A and Y57F were completely inactive to GL-7-ACA. Substituting Y126, V158, W173, W240 and L266 with an alanine resulted in decrease in catalytic efficiency. The two inflection points observed in the pH rate profile with pKa of 5.9 and 9.0 had indicated, respectively, that H309 and Y57 were involved in catalysis. Further kinetic and substrate spectrum studies had elucidated the substrate binding mechanism of J1 acylase. This study had demonstrated the previous classification of J1 acylase in the GCA classes is improper. I propose that this enzyme should be included in the alpha/beta-hydrolase superfamily evolved from the same origin of CocE and AEHs with catalytic activity towards cephalosporins. / by Yau Ming-hon. / "August 2005." / Adviser: Wang Jun. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3605. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 113-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_343675 |
| Date | January 2005 |
| Contributors | Yau, Ming Hon., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xii, 129 p. : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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