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Ansamitocins are potent antitumor agents produced by Actinosynnema pretiosum. As deduced from their structures, an N-methylation on the amide bond is required among the various modifications. The protein encoded by asm10 belongs to the SAM-dependent methyltransferase family. Through gene inactivation and complementation, asm10 was proved to be responsible for the N-methylation of ansamitocins. Asm10 is a 33.0 kDa monomer, as determined by gel filtration. By using N-desmethyl-ansamitocin P-3 as substrate, the optimal temperature and pH for Asm10 catalysis were determined to be 32 °C and 10.0, respectively. Asm10 also showed broad substrate flexibility toward other N-desmethyl-ansamycins and synthetic indolin-2-ones. Through site-directed mutagenesis, Asp154 and Leu155 of Asm10 were confirmed to be essential for its catalysis, possibly through the binding of SAM. The characterization of this unique N-methyltransferase has enriched the toolbox for engineering N-methylated derivatives from both natural and synthetic compounds; this will allow known potential drugs to be modified., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The biosynthesis of the antitumor antibiotic, ansamitocin, involves the assembly of a linear octaketide on the ansamitocin (asm) polyketide synthase (PKS), which is then cyclized to proansamitocin and further modified to the final product. In the first chain-extension step on the asm PKS, a stereocenter is generated which is then obliterated in a subsequent double-bond migration. The cryptic configuration at this stereocenter was determined by first synthesizing the two enantiomers of the intermediate diketide as their N-acetylcysteamine (SNAC) thioesters. These were then used to demonstrate that only the R enantiomer complements a 3-amino-5-hydroxybenzoic acid (AHBA) deficient mutant of Actinosynnema pretiosum to restore ansamitocin formation. The low efficiency of complementation by the diketide, compared to AHBA, is due to inefficient loading onto the PKS and not the inhibition of the enzyme. A presumed next chain-extension intermediate-the triketide with an unrearranged double bond-was also synthesized as its SNAC ester, but did not complement the AHBA(-) mutant.

Experiments on the incorporation of D- and L-[alanine-3-13C,2-15N]tryptophan into the antibiotic pyrrolnitrin in Pseudomonas aureofaciens confirmed earlier conclusions about the conversion of L-tryptophan into pyrrolnitrin. They also demonstrated that a fraction of the D isomer is incorporated without breakage of the 15N-carbon bond, consistent with the operation of a second pathway from D-tryptophan to pyrrolnitrin. Cell-free experiments confirmed the conversion of 3-(o-aminophenyl)pyrrole into aminopyrrolnitrin but failed to detect enzymatic oxidation of the latter to pyrrolnitrin.

The conversion of L-[alpha-2H]alanine in H2O and unlabeled L-alanine in 2H2O into D-alanine, under nearly irreversible conditions, with the amino acid racemase from Pseudomonas striata showed significant internal transfer of the alpha-hydrogen. This result has been interpreted as being indicative of a single base mechanism for the racemization. The relative rates of deuterium incorporation into unlabeled D- and L-methionine by the two amino acid racemases of broad substrate specificity from P. striata and Aeromonas caviae, were measured in 2H2O. The results showed a markedly different pattern, dependent upon the configuration of the initial substrate; with D-methionine as substrate deuterium is incorporated into both enantiomers at approximately the same rate, but with L-methionine as substrate deuterium is incorporated considerably faster into the D than the L enantiomer. These results argue against a single base mechanism of racemization for these enzymes and are best rationalized in terms of a double base model where only one of the bases undergoes proton (deuterium) exchange with the solvent while the amino acid is enzyme-bound. The interpretation of the earlier experiment needs to be considered in light of these results.

Using the chiral methyl group methodology, the methylation step in the biosynthesis of ergot alkaloids catalyzed by the enzyme AdoMet:dimethylallyltryptophan N-methyltransferase was found to proceed with net inversion of methyl group configuration. The enzyme thus conforms to the majority of methyltransferases studied which mediate a direct SN2 transfer of the methyl group from AdoMet to the acceptor nucleophile in a ternary enzyme substrate complex.