Purification and characterization of L-histidine aminotransferase from nikkomycin-producing Streptomyces tendae Tu901

Abstract

Cell extracts of Streptomyces tendae grown in nikkomycin production media contained an enzyme (HisAT) that transaminated L-histidine as the sole amino substrate with pyruvate as the amino group acceptor. HisAT was purified about 190-fold from the crude extract of S. tendae. The enzyme was determined by gel filtration and SDS-PAGE to be a homodimer with a subunit molecular mass of approximately 45 kDa. The aminotransferase had maximum activity at pH 7.0 and 37 OC. The enzyme was highly specific for L-histidine; pyruvate, 2-oxobutyrate, 2-oxovalerate and Zoxocaproate were used as keto acceptors to about the same extent. The reaction mechanism was ping-pong. The K, values for L-histidine and pyruvate, determined from Lineweaver-Burk plots, were 25 mM and 10 mM, respectively. Neither cell extracts of non-producing S. tendae mutants nor extracts of Streptomyces liuidans, a species that does not synthesize nikkomycins, showed transaminating activity with a narrow substrate specificity for L-histidine as the amino donor. This strongly suggests that the formation of HisAT is essential for nikkomycin production.

Identification of cellular proteins involved in nikkomycin production in Steptomyces tendae Tü901

“…The identification of protein P8 as HisAT, which has been suggested to catalyse the first reaction in the biosynthetic pathway leading to the imidazolone base of nikkomycins, allowed us to identify gene products involved in nikkomycin biosynthesis without any knowledge of fheir functions. HisAT activity strongly correlated with nikkomycin production and could not be detected in nonproducing mutants (Roos etai, 1992: Roos andBormann, 1993). The W-terminus of purified HisAT lacked two amino acid residues, in contrast to that of protein P8.…”