title: Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues.
abstract: The class II PHA (polyhydroxyalkanoate) synthases [PHA(MCL) synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHA(MCL)s using CoA thioesters of medium-chain-length 3-hydroxy fatty acids (C6-C14) as a substrate. Only recently PHA(MCL) synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHA(MCL) synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the alpha/beta-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the alpha/beta-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved gamma-turns of the alpha/beta-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue. In the threading model this residue was found to be surface-exposed. No evidence for post-translational modification by 4-phosphopantetheine was obtained. Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the alpha/beta-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.
authors.affiliation: Institut für Molekulare Mikrobiologie und Biotechnologie der Westfälischen Wilhelms-Universität Münster, Corrensstrasse 3, 48149 Münster, Germany.
authors.forename: Amro A
authors.initials: AA
authors.lastname: Amara
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004851:Epoxide Hydrolases / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000737:chemistry; D011091:Polyesters / Q000138:chemical synthesis*; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D012694:Serine / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D011091:Polyesters; D012694:Serine; D010204:Pantetheine; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; D004851:Epoxide Hydrolases; D003545:Cysteine; C003129:4'-phosphopantetheine