Partial purification and characterization of a C-methyltransferase from streptonigrin-producing Streptomyces flocculus

Abstract

A C-methyltransferase which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the C-3 position of the aliphatic side chain of L-tryptophan resulting in formation of {dollar}\beta{dollar}-methyl tryptophan, has been isolated from streptonigrin-producing Streptomyces flocculus. The enzyme catalyzes the first step in streptonigrin biosynthesis and is postulated to have a regulatory role in the pathway. The enzyme has been purified 217-fold by ammonium sulfate fractionation, followed by sequential gel filtration through Sephadex G-150 and Sephadex G-100 SF columns. Attempts at further purification have been hindered by very active proteases which co-purify with the enzyme. Protease inhibitors PMSF, pepstatin A, leupeptin, and trypsin inhibitor have failed to inactivate the protease activity. Based on comparison to reference proteins, the C-methyltransferase was estimated to have a molecular weight of 40,000 by Sephadex G-150 gel filtration. A narrow pH optimum of 7.5-8.0 was determined for the enzyme. The Sephadex G-100 SF fraction was highly unstable, losing 90 {dollar}\pm{dollar} 6% of its activity after 12 hours at 4{dollar}\sp\circ{dollar}C. S-Adenosyl-L-methionine and L-cysteine have been found to stabilize activity in the purified fractions. The enzyme is inhibited by sulfhydryl binding reagents, but no such inhibition is observed in the presence of substrate, suggesting an essential {dollar}-{dollar}SH group at or near the active site. Inhibition by carbonyl reagents was exhibited by the C-methyltransferase. Tritiated sodium cyanoborohydride treatment of the Sephadex G-100 SF fraction resulted in tritium incorporation and a concomitant 36 {dollar}\pm{dollar} 1% inactivation of the enzyme. These combined data led to the hypothesis that pyridoxal-5{dollar}\sp\prime{dollar}-phosphate may be involved as a cofactor in the C-methyltransferase. An enzymatic mechanism is proposed, and several studies related to this mechanism are presented.