Aromatic C-Methyltransferases with Antipodal Stereoselectivity for Structurally Diverse Phenolic Amino Acids Catalyze the Methylation Step in the Biosynthesis of the Actinomycin Chromophore

The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus harbors two paralogous genes, acmI and acmL, encoding methyltransferases. To unveil their suspected role in the formation of 3-hydroxy-4-methyl-anthranilic acid (4-MHA), the building block of the actinomycin chromophore, each gene was expressed in Escherichia coli. Testing the resulting ∼40 kDa His6-tagged proteins with compounds of biogenetic relevance as substrates and S-adenosyl-l-methionine revealed that each exclusively methylated 3-hydroxykynurenine (3-HK) with formation of 3-hydroxy-4-methylkynurenine (4-MHK) identified by its in vitro conversion to 4-MHA with hydroxykynureninase. AcmI and AcmL methylate also hydroxyphenyl-amino propanoic acids such as p-tyrosine, m-tyrosine, or 3,4-dihydroxy-l-phenylalanine (DOPA) but at a lower rate than 3-HK. The presence of the α-amino group was necessary for substrate recognition. Phenolic acids with shorter chains such as 4-hydoxyphenyl-l-glycine (HPG), 3-hydroxybenzoic acid (3-HB), or 3-h...