ABSTRACTWe have recently cloned ad-cycloserine (DCS) biosynthetic gene cluster that consists of 10 genes, designateddcsA∼dcsJ, fromStreptomyces lavendulaeATCC 11924 (16). In the predicted pathway of hydroxyurea (HU) formation in DCS biosynthesis,l-arginine (L-Arg) must first be hydroxylated, prior to the hydrolysis ofNω-hydroxy-l-arginine (NHA) by DcsB, an arginase homolog. The hydroxylation of L-Arg is known to be catalyzed by nitric oxide synthase (NOS). In this study, to verify the supply route of HU, we created adcsB-disrupted mutant, ΔdcsB. While the mutant lost DCS productivity, its productivity was restored by complementation ofdcsB, and also by the addition of HU but not NHA, suggesting that HU is supplied by DcsB. A NOS-encoding gene,nos, fromS. lavendulaechromosome was cloned, to create anos-disrupted mutant. However, the mutant maintained the DCS productivity, suggesting that NOS is not necessary for DCS biosynthesis. To clarify the identity of an enzyme necessary for NHA formation, adcsA-disrupted mutant, designated ΔdcsA, was also created. The mutant lost DCS productivity, whereas the DCS productivity was restored by complementation ofdcsA. The addition of NHA to the culture medium of ΔdcsAmutant was also effective to restore DCS production. These results indicate that thedcsAgene product, DcsA, is an enzyme essential to generate NHA as a precursor in the DCS biosynthetic pathway. Spectroscopic analyses of the recombinant DcsA revealed that it is a heme protein, supporting an idea that DcsA is an enzyme catalyzing hydroxylation.