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Abstract The thiamine diphosphate (ThDP)‐binding motif, characterized by the canonical GDG(X) 24‐27 N sequence, is highly conserved among ThDP‐dependent enzymes. We investigated a ThDP‐dependent lyase (JanthE from Janthinobacterium sp. HH01 ) with an unusual cysteine (C458) replacing the first glycine of this motif. JanthE exhibits a high substrate promiscuity and accepts long aliphatic α ‐keto acids as donors. Sterically hindered aromatic aldehydes or non‐activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Å reveals that C458 is not primarily involved in cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. In addition, we have determined the long‐sought genuine tetrahedral intermediates formed with pyruvate and 2‐oxobutyrate in the pre‐decarboxylation states and deciphered the atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP‐binding motif and unlock a family of lyases that can perform valuable carboligation reactions.
Lanza et al. (Fri,) studied this question.