Abstract
MbtH-like proteins consist of ∼70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes.
Highlights
L-Tyrosine adenylation is a key step in aminocoumarin antibiotic and vancomycin biosynthesis
In vivo studies have suggested that the formation of the aminocoumarin moiety of clorobiocin requires an intact mbtH-like gene, but the identical reaction sequence in novobiocin biosynthesis does not [2]
To exclude the possibility that YbdZ would copurify with the expressed adenylating enzymes and interfere with subsequent investigations, we deleted the ybdZ gene from the E. coli expression host utilizing the same Red/ET-mediated recombination strategy as described in a previous study of our group [11]
Summary
L-Tyrosine adenylation is a key step in aminocoumarin antibiotic and vancomycin biosynthesis. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. Of the five adenylation domains that activate the different amino acids required for capreomycin biosynthesis, three were dependent on the presence of the MbtH-like protein CmnN, whereas the two others were not. In vivo studies have suggested that the formation of the aminocoumarin moiety of clorobiocin requires an intact mbtH-like gene, but the identical reaction sequence in novobiocin biosynthesis does not [2]. Given the facts that the tyrosine-adenylating enzyme NovH has the same size (600 amino acids) as CloH and that both proteins share 83% identity in their amino acid sequences (supplemental Fig. S1), this difference in the requirement for MbtH-like proteins is puzzling. We decided to express the tyrosine-adenylating enzymes NovH, CloH, SimH, and Pzca361.18 as well as the cognate MbtH-like proteins and to biochemically investigate these enzymes and the complexes formed by them
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