Structures and Protein Engineering of the α-Keto Acid C-Methyltransferases SgvM and MrsA for Rational Substrate Transfer.

S-adenosyl-l-methionine-dependent methyltransferases (MTs) are involved in the C-methylation of a variety of natural products. The MTs SgvM from Streptomyces griseoviridis and MrsA from Pseudomonas syringae pv. syringae catalyze the methylation of the ß-carbon atom of α-keto acids in the biosynthesis of the antibiotic natural products viridogrisein and 3-methylarginine, respectively. MrsA shows high substrate selectivity for 5-guanidino-2-oxovalerate, while other α-keto acids, such as the SgvM substrates 4-methyl-2-oxovalerate, 2-oxovalerate, and phenylpyruvate, are not accepted. Here we report the crystal structures of SgvM and MrsA in the apo form and bound with substrate or S-adenosyl-l-methionine. By investigating key residues for substrate recognition in the active sites of both enzymes and engineering MrsA by site-directed mutagenesis, the substrate range of MrsA was extended to accept α-keto acid substrates of SgvM with uncharged and lipophilic ß-residues. Our results showcase the transfer of the substrate scope of α-keto acid MTs from different biosynthetic pathways by rational design.

Substrate recognition and mechanism revealed by ligand-bound polyphosphate kinase 2 structures.

Inorganic polyphosphate is a ubiquitous, linear biopolymer built of up to thousands of phosphate residues that are linked by energy-rich phosphoanhydride bonds. Polyphosphate kinases of the family 2 (PPK2) use polyphosphate to catalyze the reversible phosphorylation of nucleotide phosphates and are highly relevant as targets for new pharmaceutical compounds and as biocatalysts for cofactor regeneration. PPK2s can be classified based on their preference for nucleoside mono- or diphosphates or both. The detailed mechanism of PPK2s and the molecular basis for their substrate preference is unclear, which is mainly due to the lack of high-resolution structures with substrates or substrate analogs. Here, we report the structural analysis and comparison of a class I PPK2 (ADP-phosphorylating) and a class III PPK2 (AMP- and ADP-phosphorylating), both complexed with polyphosphate and/or nucleotide substrates. Together with complementary biochemical analyses, these define the molecular basis of nucleotide specificity and are consistent with a Mg2+ catalyzed in-line phosphoryl transfer mechanism. This mechanistic insight will guide the development of PPK2 inhibitors as potential antibacterials or genetically modified PPK2s that phosphorylate alternative substrates.