TAILIEUCHUNG - Báo cáo khoa học: Biochemical characterization of MelJ and MelK Myxobacterial enzymes that transform an amide into a methyl ester

Melithiazol and myxothiazol are two myxobacterial metabolites that are highly efficient electron transport inhibitors of the respiratory chain. MelJ and MelK encoded in the melithiazol biosynthetic gene cluster were recently shown to be involved in the formation of the methyl ester from a hypothetical amide intermediate. | ễFEBS Journal Biochemical characterization of MelJ and MelK Myxobacterial enzymes that transform an amide into a methyl ester I. Muller and R. Muller PharmaceuticalBiotechnology Saarland University Saarbrucken Germany Keywords myxothiazol melithiazol hydrolase methyl transferase enzyme characterization Correspondence R. Muller PharmaceuticalBiotechnology Saarland University . Box 151150 D-66041 Saarbrucken Germany Fax 49 681 302 5473 Tel 49 681 302 5474 E-mail rom@ Received 10 May 2006 revised 13 June 2006 accepted 16 June 2006 doi Melithiazol and myxothiazol are two myxobacterial metabolites that are highly efficient electron transport inhibitors of the respiratory chain. MelJ and MelK encoded in the melithiazol biosynthetic gene cluster were recently shown to be involved in the formation of the methyl ester from a hypothetical amide intermediate. In vivo studies suggest that the structurally highly similar amide myxothiazol A can be used as a substrate mimic of the hypothetical melithiazol amide to characterize the hydrolase MelJ. Both enzymes were produced in Escherichia coli as intein chitin fusion proteins and were purified using affinity chromatography. MelJ was found to catalyse the conversion of the amide myxothiazol to free myxothiazol acid. The formerly unknown myxothiazol acid was purified and used as a substrate for the methyl transferase MelK which methylates the compound using S-adenosyl-methionine as cosubstrate. Sequence analyses suggest that MelJ and MelK are members of the amidase signature family and of a new subclass of methyltransferases respectively. Kinetic analyses point at a very high substrate specificity for both enzymes. Furthermore the in vitro reconstitution of a unique mechanism of methyl ester formation found in myxo-bacteria is reported. Myxobacteria produce a wide range of biologically active substances that are of interest to the pharmaceutical and agrochemical industries 1 . .

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