Mutational analysis and characterization of nocardicin C-9' epimerase.
The biosynthetic gene cluster for the nocardicin A producer Nocardia uniformis subsp. tsuyamanensis ATCC 21806 was recently identified. Nocardicin A is the most potent of a series of monocyclic beta-lactam antibiotics produced by this organism. Its activity has been attributed to a syn-configured oxime moiety and a d-homoseryl side chain attached through an unusual ether linkage to the core nocardicin framework. Notably present in the nocardicin biosynthetic gene cluster is nocJ, encoding a protein with sequence similarity to the pyridoxal 5'-phosphate (PLP)-dependent 1-aminocyclopropane-1-carboxylic acid deaminases. Insertional mutagenesis of nocJ abolished nocardicin A production, while the l-homoseryl isomer, isonocardicin A, was still observed. Expression of the disrupted nocJ gene in trans was sufficient to restore production of nocardicin A in the disruption mutant. Heterologous expression, purification, and in vitro characterization of NocJ by UV spectroscopy, cofactor reduction, chiral HPLC analysis of the products and their exchange behavior in deuterium oxide led to confirmation of its role as the PLP-dependent nocardicin C-9' epimerase responsible for interconversion of the nocardicin homoseryl side chain in both nocardicin A with isonocardicin A, and nocardicin C with isonocardicin C. NocJ is the first member of a new class of beta-lactam aminoacyl side chain epimerases, the first two classes being the evolutionarily distinct prokaryotic PLP-dependent isopenicillin N epimerase and the fungal isopenicillin N epimerase two protein system.
Kelly
Wendy L
WL
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Townsend
Craig A
CA
D000591:Amino Acid Isomerases / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry; D001681:Biological Assay; D015139:Blotting, Southern; D001894:Borohydrides / Q000494:pharmacology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004252:DNA Mutational Analysis; D003903:Deuterium / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D007700:Kinetics; D007769:Lactams / Q000737:chemistry* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D005810:Multigene Family; D009615:Nocardia / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D020033:Protein Isoforms; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013997:Time Factors; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D001894:Borohydrides; D007769:Lactams; D020033:Protein Isoforms; C027318:nocardicin; C025364:sodium borohydride; D004247:DNA; D003903:Deuterium; D019998:Racemases and Epimerases; D000591:Amino Acid Isomerases; C040886:isopenicillin N epimerase; C492428:nocardicin C-9' epimerase, Nocardia uniformis