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Eur J Biochem. 1995 May 15;230(1):119-26.

Arrangement of catalytic sites in the multifunctional enzyme enniatin synthetase.

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1
Institut für Biochemie und Molekulare Biologie, Technische Universität Berlin, Germany.

Abstract

Enniatin synthetase is an N-methyl peptide synthetase comprising 3131 amino acids. Catalytic sites of the 347-kDa multifunctional enzyme were mapped by N-terminal sequencing of substrate affinity-labelled enzyme fragments formed by proteolysis, and functional studies of purified enniatin synthetase fragments. An N-terminal 200-kDa fragment containing the cofactor 4'-phosphopantetheine was able to activate D-hydroxyisovaleric acid (D-HOiVl) as a thioester. The N-termini of two [14C]HOiVl-labelled enzyme fragments could be assigned to amino acid position 429 within the N-terminal conserved enniatin synthetase portion named EA. This portion of about 600 amino acids shares high similarity to microbial peptide synthetase regions. A 68-kDa L-[14C]Val-labelled enniatin synthetase fragment was localized at amino acid position 2294 within the second C-terminal conserved protein portion EB. Additionally enniatin synthetase was labelled with isovaleryl-L-[14C]Val, an analogue of the D-hydroxyisovaleryl-L-Val intermediate in enniatin biosynthesis. The N-terminus of a 30-kDa isovaleryl-L-[14C]Val-labelled enniatin synthetase fragment was mapped in a C-terminal segment of the protein portion EA. The same N-terminal sequence was obtained from a 60-kDa enniatin synthetase fragment modified with [3H]beta Ala, a constituent of the cofactor 4'-phosphopantetheine. This indicates the presence of the cofactor in this protein fragment. Localization of the methyltransferase function of enniatin synthetase in an amino acid portion integrated into region EB was achieved by N-terminal sequencing of a photolabelled S-[methyl-14C]adenosyl methionine 45-kDa fragment and the identification of a photolabelled peptide Asn-Leu-Asn-Pro-Gly-Leu-Asn-Ser-Tyr.

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