Structural studies reveal flexible roof of active site responsible for ω-transaminase CrmG overcoming by-product inhibition

Abstract

Amine compounds biosynthesis using ω-transaminases has received considerable attention in the pharmaceutical industry. However, the application of ω-transaminases was hampered by the fundamental challenge of severe by-product inhibition. Here, we report that ωtransaminase CrmG from Actinoalloteichus cyanogriseus WH1-2216-6 is insensitive to inhibition from by-product α-ketoglutarate or pyruvate. Combined with structural and QM/MM studies, we establish the detailed catalytic mechanism for CrmG. Our structural and biochemical studies reveal that the roof of the active site in PMP-bound CrmG is flexible, which will facilitate the PMP or by-product to dissociate from PMP-bound CrmG. Our results also show that amino acceptor caerulomycin M (CRM M), but not α-ketoglutarate or pyruvate, can form strong interactions with the roof of the active site in PMP-bound CrmG. Based on our results, we propose that the flexible roof of the active site in PMP-bound CrmG may facilitate CrmG to overcome inhibition from the by-product.

Stereoselective amino acid synthesis by synergistic photoredox-pyridoxal radical biocatalysis

“…5; see the supplementary materials for computational details). Consistent with previous computational studies on related PLP-dependent enzymes ( 57 , 58 ), the conversion between the internal aldimine and external aldimine 7 requires a low activation barrier (fig. S16).…”

Kinetic Characterization and Catalytic Mechanism of N-Acetylornithine Aminotransferase Encoded by slr1022 Gene from Synechocystis sp. PCC6803

“…After the addition of AcOrn to recombinant Slr1022, which reacted for 30 min, the absorbance at 410 nm decreased and a new absorption peak at 340 nm appeared, which meant that PLP was converted to PMP in the first half reaction ( Figure 5 and Figure S4 ). Due to the short reaction time (30 min), the complete conversion of PLP to PMP was not observed as that of CrmG protein reported by Xu et al [ 22 ].…”

Deracemization of Racemic Amine Using ω-Transaminase and a Nickel-Based Nanocatalyst

“…The use of amine transaminase, also known as ω-transaminase (ω-TA), and a pyridoxal-5′-phosphate (PLP)-dependent enzyme to synthesize chiral amines is both a feasible and effective method . Although ω-TA-promoted asymmetric synthesis can theoretically provide an ideal yield of the desired chiral amines with satisfying ees during transamination, as reversible reactions, it suffers from equilibrium issuesespecially, in some asymmetric amine syntheses, the equilibrium can be far on the side of the substrates . The problems relating to equilibrium displacement have attracted considerable attention.…”

“…12 Although ω-TA-promoted asymmetric synthesis can theoretically provide an ideal yield of the desired chiral amines with satisfying ees during transamination, as reversible reactions, it suffers from equilibrium issues�especially, in some asymmetric amine syntheses, the equilibrium can be far on the side of the substrates. 10 The problems relating to equilibrium displacement have attracted considerable attention. Several studies have tackled unfavored equilibria through (i) employing (large) excess of cosubstrate (amino donor); 13 (ii) removal of product or coproduct (amino acceptor) through physical measures; 14,15 (iii) multi-enzyme cascades, where the equilibria are replaced with in situ removal of coproduct by combining expensive cofactor-dependent enzymes; 16 (iv) substrate engineering to lower the expense on coproduct removals; 17 and (v) triple-superposition driving force using amino donor regeneration.…”

“…Nevertheless, requisite chiral amines are scarce in nature. − Large-scale preparation of chiral amines is mainly carried out by metal catalyst-based asymmetric hydrogenation of enamides (i.e., obtained from related ketone precursors). In the past decade, various methods have been developed for the direct conversion of ketones into chiral amines − and the asymmetrical hydrogenation of oximes and CN bond-bearing intermediates (i.e., imines) into chiral amines . However, most of these methods require harsh reaction conditions leading to low efficiency and high environmental impact, resulting in low product enantiomeric excess (ee).…”