An investigation into the enzymes required for biosynthesis of the beta-lactam antibiotic nocardicin A.

摘要

The nocardicins are monocyclic beta-lactam antibiotics produced by the actinomycete Nocardia uniformis subsp, tsuyamanensis ATCC 21806 (N. uniformis). Two of the key features of nocardicin A required for maximal antimicrobial potency are the syn-oxime moiety at C-2' and the D-configuration at C-9' of the homoseryl side chain. The homoseryl side chain itself is derived from S-adenosyl-L-methionine. The peptide core of the nocardicin series originates from two L-4-hydroxyphenylglycine units and one molecule of L-serine. The nocardicin A biosynthetic gene cluster was recently identified. Three of these genes were examined with respect to their role in nocardicin A biosynthesis: nocJ, nocK, and nocL. This was accomplished by insertional mutagensis of each gene of interest and by direct biochemical assays based on a rational prediction of function. To accomplish the disruption mutagenesis, the development of a transformation protocol for N. uniformis was necessary, as there was no literature precedent for genetic manipulations with this organism.; The following mutants were constructed: nocJ::apra R N. uniformis, nocK::apraR N. uniformis, and nocL::apraR N. uniformis. Nocardicin A production was unaltered in nocK::apraR N. uniformis , implying a nonessential role, if any, for this gene product in nocardicin A biosynthesis. Both the nocJ and the nocL mutant strains lost the ability to produce nocardicin A, accumulating isonocardicin A and nocardicin C, respectively. Antibiotic production was restored by in trans complementation of the appropriate gene, confirming the loss of nocardicin A from the fermentation broth was solely due to disruption of the target gene.; Both NocJ and NocL were heterologously expressed as the hexahistidine tag fusion proteins and purified to apparent homogeneity. NocJ was found to require the PLP cofactor for activity, and to catalyze epimerization at C-9 ' of the homoseryl side chain present in nocardicin C and nocardicin A. NocL was shown to be a cytochrome P450 enzyme responsible for N-oxygenation of the nocardicin C primary amine to generate the oxime present in nocardicin A. NocL is unique as it is the first prokaryotic cytochrome P450 capable of oxime formation, a rare reaction in natural product biosynthesis.