11. Structural insights into two thiamine diphosphate-dependent enzymes and their synthetic applications in carbon–carbon linkage reactions

The α-hydroxy-β-keto acid synthases arethiamine diphosphate-dependent enzymes catalysing carbon–carbon linkagereactions in the biosynthesis of primary metabolites and various secondarymetabolites. However, the substrate selectivity and catalytic stereoselectivityof α-hydroxy-β-keto acid synthases are poorly understood, greatly hinderingtheir synthetic application in generating diverse carbon frameworks. We herereport the discovery of two new α-hydroxy-β-keto acid synthases CsmA and BbmA, whichshow different substrate selectivities in catalysing carbon–carbon couplingreactions between two β-keto acids. Four crystal structures of CsmA or BbmAcomplexed with thiamine diphosphate and their substrates were determined,clearly revealing their structural bases of substrate selectivity and catalyticstereoselectivity. Substrate scope expansion enables us to generate 120α-hydroxy-β-keto acids together with 240 NaBH4-reduction products. Furthermore,we applied CsmA and BbmA into enzymatic total synthesis, generating 36γ-butyrolactone-containing furanolides. These results provide new structuralinsights into the catalyses of α-hydroxy-β-keto acid synthases and highlighttheir great potential in carboligation catalysis and synthetic applications.