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

Biochemical Characterization Of Melj And Melk: Myxobacterial Enzymes That Transform An Amide Into A Methyl Ester

Authors: I. Müller and R. Müller

Field: Pharmaceutical Biotechnology, Saarland University, Saarbrücken, Germany

Document Content:
This study investigates the biochemical characterization of two myxobacterial enzymes, MelJ and MelK, involved in the transformation of a hypothetical amide intermediate into a methyl ester. These enzymes are encoded within the melithiazol biosynthetic gene cluster. The research details the heterologous expression and purification of MelJ and MelK in Escherichia coli. Experimental findings demonstrate that MelJ catalyzes the conversion of the amide myxothiazol A to myxothiazol acid, while MelK methylates this acid to form the methyl ester myxothiazol Z, utilizing S-adenosyl-methionine as a cosubstrate. Sequence analysis suggests MelJ belongs to the amidase signature family, and MelK represents a novel subclass of methyltransferases. Kinetic analyses indicate high substrate specificity for both enzymes. The study also reports on the successful in vitro reconstitution of the unique mechanism of methyl ester formation observed in myxobacteria.

Detailed Table of Contents:

• Keywords
• Correspondence
• Abbreviations
• Abstract
• Results
• Properties of MelJ
• Properties of MelK
• Discussion
• In vitro reconstitution of methyl ester formation
• Preparation of the Melk substrate myxothiazol acid
• Enzyme assays for MelJ
• Assays employing alternative substrates for MelJ
• Kinetic analysis
• Acknowledgements
• References
• Experimental procedures
• Strains and media
• Gene amplification and expression
• Expression and purification of Melk and MelJ
• Western blot analysis
• In vitro reconstitution of methyl ester formation
• Preparation of the Melk substrate myxothiazol acid
• Characterization of MelJ
• Characterization of MelK