PMID:15663941

Li, C, Montgomery, MG, Mohammed, F, Li, JJ, Wood, SP and Bugg, TD (2005) Catalytic mechanism of C-C hydrolase MhpC from Escherichia coli: kinetic analysis of His263 and Ser110 site-directed mutants.J. Mol. Biol. 346:241-51

Abstract

C-C hydrolase MhpC (2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase) from Escherichia coli catalyses the hydrolytic C-C cleavage of the meta-ring fission product on the phenylpropionic acid catabolic pathway. The crystal structure of E. coli MhpC has revealed a number of active-site amino acid residues that may participate in catalysis. Site-directed mutants of His263, Ser110, His114, and Ser40 have been analysed using steady-state and stopped-flow kinetics. Mutants H263A, S110A and S110G show 10(4)-fold reduced catalytic efficiency, but still retain catalytic activity for C-C cleavage. Two distinct steps are observed by stopped-flow UV/Vis spectrophotometry, corresponding to ketonisation and C-C cleavage: H263A exhibits very slow ketonisation and C-C cleavage, whereas S110A and S110G exhibit fast ketonisation, an intermediate phase, and slow C-C cleavage. H114A shows only twofold-reduced catalytic efficiency, ruling out a catalytic role, but shows a fivefold-reduced K(M) for the natural substrate, and an ability to process an aryl-containing substrate, implying a role for His114 in positioning of the substrate. S40A shows only twofold-reduced catalytic efficiency, but shows a very fast (500 s(-1)) interconversion of dienol (317 nm) to dienolate (394 nm) forms of the substrate, indicating that the enzyme accepts the dienol form of the substrate. These data imply that His263 is responsible for both ketonisation of the substrate and for deprotonation of water for C-C cleavage, a novel catalytic role in a serine hydrolase. Ser110 has an important but non-essential role in catalysis, which appears not to be to act as a nucleophile. A catalytic mechanism is proposed involving stabilisation of reactive intermediates and activation of a nucleophilic water molecule by Ser110.

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Annotations

Gene product	Qualifier	GO ID	GO term name	Evidence Code	with/from	Aspect	Notes	Status
MhpC		GO:0016787	hydrolase activity	IMP: Inferred from Mutant Phenotype		F	Purified MhpC and several mutants were assayed for 2-hydroxy-6-keto-nona-1,9-dioic acid 5,6-hydrolase activity by measuring fluorescence at 394nm, thereby monitoring the disappearance of the substrate for MhpC, 2-Hydroxy-6-keto-nona-2,4-diene-1,9-dioic acid.	complete
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