Demethylmacrocin

The S-adenosyl-l-methionine-dependent O-methyltransferases TylE and TylF catalyze the last two methylation reactions in the tylosin biosynthetic pathway of Streptomyces fradiae. It has long been known that the TylE-catalyzed C2‴-O-methylation of the 6-deoxy-d-allose bound to demethylmacrocin or demethyllactenocin precedes the TylF-catalyzed C3‴-O-methylation of the d-javose (C2‴-O-methylated 6-deoxy-d-allose) attached to macrocin or lactenocin. This study reveals the unexpected substrate promiscuity of TylE and TylF responsible for the biosynthesis of d-mycinose (C3‴-O-methylated d-javose) in tylosin through the identification of a new minor intermediate 2‴-O-demethyldesmycosin (2; 3‴-methyl-demethyllactenocin), which lacks a 2‴-O-methyl group on the mycinose moiety of desmycosin, along with 2‴-O-demethyltylosin (1; 3‴-methyl-demethylmacrocin) that was previously detected from the S. fradiae mutant containing a mutation in the tylE gene. These results unveil the unique substrate flexibility of TylE and TylF and demonstrate their potential for the engineered biosynthesis of novel glycosylated macrolide derivatives.

S-Adenosyl-L-methionine:demethylmacrocin O-methyltransferase catalyzes the conversion of demethylmacrocin to macrocin as the penultimate step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was purified to electrophoretic homogeneity by a conventional chromatographic procedure. The purified enzyme appears to be trimeric with a molecular weight of 122,000-126,000 and a subunit size of 42,000. Its isoelectric point was 6.0. The enzyme required Mg2+ for maximal activity and was catalytically optimal at pH 7.8-8.5 and 42 degrees C. The O-methyltransferase catalyzed conversion of demethylmacrocin to macrocin at a stoichiometric ratio of 1:1. The O-methyltransferase also mediated conversion of demethyllactenocin----lactenocin. The corresponding Vmax/Km ratios for the two analogous conversions varied only slightly. Both enzymic conversions were susceptible to an extensive and identical range of metabolic inhibitions. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions are consistent with Ordered Bi Bi as the reaction mechanism of demethylmacrocin O-methyltransferase. Except for an identical kinetic mechanism, demethylmacrocin O-methyltransferase can be readily differentiated from macrocin O-methyltransferase by its physical and catalytic properties as well as metabolic inhibitions.

The case studies focus on two types of enzyme applications for pharmaceutical development. Demethylmacrocin O-methyltransferase, macrocin O-methyltransferase (both putatively rate-limiting) and tylosin reductase were purified from Streptomyces fradiae, characterized and the genes manipulated for increasing tylosin biosynthesis in S. fradiae. The rate-limiting enzyme, deacetoxycephalosporin C (DAOC) synthase/hydroxylase (expandase/ hydroxylase), was purified from Cephalosporium acremonium, its gene over-expressed, and cephalosporin C biosynthesis improved in C. acremonium. Also, heterologous expression of penicillin N epimerase and DAOC synthase (expandase) genes of Streptomyces clavuligerus in Penicillium chrysogenum permitted DAOC production in the fungal strain. Second, serine hydroxymethyltransferase of Escherichia coli and phthalyl amidase of Xanthobacter agilis were employed in chemo-enzymatic synthesis of carbacephem. Similarly, echinocandin B deacylase of Actinoplanes utahensis was used in the second-type synthesis of the ECB antifungal agent.