The thiamine diphosphate-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) catalyzes a Stetter-like 1,4-addition of α-ketoglutarate to isochorismate in the biosynthesis of menaquinone (vitamin K). Here, we describe the carboligation potential of MenD from Bacillus subtilis (BsMenD) for the nonphysiological 1,2-addition of decarboxylated α-ketoglutarate (succinylsemialdehyde) and various benzaldehyde derivatives. Furthermore, we engineer BsMenD variants for the enantiocomplementary asymmetric synthesis of functionalized α-hydroxy ketones. Wild type BsMenD shows an excellent chemo- as well as high (R)-selectivity for the carboligation of α-ketoglutarate as the donor, and different benzaldehyde derivatives as acceptor yielding (R)-α-hydroxy ketones with up to >99 % ee. By engineering (S)-selective BsMenD variants, based on the recently developed S-pocket concept, we provide access to most of the corresponding (S)-α-hydroxy ketones with up to 98 % ee. In particular, benzaldehyde and meta-substituted derivatives were converted with high enantioselectivities (ee of 91–98 % (S)). The significantly higher (S)-selectivity of BsMenD variants than recently published MenD variants from Escherichia coli, could be attributed to a second-shell residue next to the S-pocket. A glycine residue, adjacent to the major S-pocket residues I476 and F477 (standard numbering), is assumed to result in higher structural flexibility in the S-pocket region of BsMenD, which in turn could result in improved stabilization of the antiparallel orientation of the acceptor.
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