N-methylation of the amide bond by methyltransferase asm10 in ansamitocin biosynthesis

Abstract

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.

Figure 1

N-desmethylansamitocins, ansamitocins and the asm biosynthetic gene cluster. A) Structures of N-desmethylansamitocins (PNDs) and ansamitocins (APs) with N-methyl moiety highlighted. B) The ansamitocin biosynthetic gene cluster. The N-methyltransferase gene asm10 is indicated by a circle in Cluster I. AHBA, 3-amino-5-hydroxybenzoic acid; MM-ACP, methoxy-malonyl-ACP.

Figure 2

Inactivation of asm10 and complementation of the mutant BLQ12. A) Schematic construction of the asm10 mutant. The shuttle plasmid pJTU654 was sequentially treated by digestion with KpnI, erased with Klenow, and religated with T4 ligase. K, KpnI; B, BamHI. B) Gel electrophoresis of the PCR amplified fragments. The left gel showed the PCR products using total DNA of the wild-type or the mutant BLQ12 as templates. The right gel showed the two PCR products digested with KpnI. Whereas the PCR product of the wild-type could be cleaved into 670-bp and 210-bp fragments, the PCR products of BLQ12 kept the original size of 880 bp after KpnI digestion. C) HPLC profiles of the wild-type, asm10 mutant BLQ12, pIB139 vector-complemented strain KW1, and asm10-complemented strain KW2 cultured on solid ISP2 medium. AP, ansamitocin; PND, N-desmethyl-ansamitocin; AGP, ansamitocinoside.

Figure 3

Catalytic properties of Asm10. A) A representative Coomassie Brilliant Blue-stained SDS-PAGE gel loaded with molecular weight markers (lane 1), total proteins (lane 2), soluble proteins (lane 3), and purified Asm10 (lane 4). B) Temperature optimum for the N-methylation of PND-3 catalyzed by Asm10. C) pH optimum for the N-methylation of PND-3 catalyzed by Asm10. Asm10 was assayed in 50 mM Tris-HCl buffer ranging from pH 5.5 to 13.0. D) pH experiment (pH 9, 10, 11) in the linear reaction phase.

Figure 4

Kinetic characterization of Asm10. A) The initial reaction rate constructed with different concentration of PND-3. B) The initial reaction rate constructed with different concentration of SAM.

Figure 5

Substrate specificity of Asm10.

Figure 6

In vitro N-methylation of PNDs by Asm10 and its mutated proteins. A) HPLC analysis of the methylation of PND-3 by Asm10 and its mutated proteins. B) Comparison of the kinetics of Asm10 and the L155I mutated protein.