The ornithine carbamoyltransferases (OTCases) from the β-lactam-producing actinomycetes Streptomyces clavuligerus and Nocardia lactamdurans have been purified to near-homogeneity by Δ-N-phosphonoacetylornithine-Sepharose 4B affinity chromatography. The S. clavuligerus and N. lactamdurans OTCase monomers had a molecular mass of 37 kDa. The native OTCases of S. clavuligerus, N. lactamdurans and Streptomyces coelicolor had molecular masses of 248, 251 and 247 kDa respectively, which correspond to a hexameric structure. The apparent Km values for ornithine and carbamoylphosphate of the S. clavuligerus enzyme were respectively 2.3 and 6.0 mM at pH 8.0. The enzyme showed a reverse activity on citrulline and used lysine and putrescine as substrates. The hexameric complex showed coupled arginase–OTCase activities and was able to convert arginine into citrulline in a carbamoylphosphate-dependent manner. The requirement for carbamoylphosphate might prevent the arginase–OTCase complex from carrying out a futile cycle of arginine biosynthesis and degradation.

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