• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.798-803
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• 2001

Peptide synthetases produced from various microorganisms are multifunctional enzyme complexes and their substrates are recognized and activated by adenylation domains. To identify the substrate specificity of the peptide synthetase isolated from Bacillus subtilis 713, known to produce an antifungal peptide, two adenylation domains containing the minimal functional portion were expressed and purified. ATP-ppi exchange experiments and kinetic studies revealed that the two adenylation enzymes had a substrate specificity to $_{L}-lysine{\;}and{\;}_{L}-tryptophan$, respectively. In addition, based on a signature sequence comparison, the substrate of the third domain was predicted to be L-glutamic acid. These results suggest that this peptide synthetase is novel because there has been no previous report on a peptide synthetase that uses $_{L}-lysine,{\;}_{L}-tryptophan,{\;}and{\;}_{L}-glutamic$ acid as substrates in that order.

• Microbiology and Biotechnology Letters
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• v.24 no.1
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• pp.67-71
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• 1996

The plasmid pK8 was constructed to verify the existence of an adenylate domain in peptide synthetase by using pGC12. 1.2 kb fragment, coding tyrocidine synthetase 1 (123 kDa) was deleted, and 79.6 kDa one was expressed in Escherichia coli XL1-blue. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over expressed synthetase. ATP-[$^{32}P$]PPi exchange reaction was measured for the enzyme assay.