Evidence of Diradicals Involved in the Yeast Transketolase Catalyzed Keto‐Transferring Reactions

• Published in: Chembiochem : a European journal of chemical biology Vol. 19; no. 22; pp. 2395 - 2402
• Main Authors: Hsu, Ning‐Shian, Wang, Yung‐Lin, Lin, Kuan‐Hung, Chang, Chi‐Fon, Ke, Shyue‐Chu, Lyu, Syue‐Yi, Hsu, Li‐Jen, Li, Yi‐Shan, Chen, Sheng‐Chia, Wang, Kuei‐Chen, Li, Tsung‐Lin
• Format: Journal Article
• Language: English
• Published: WEINHEIM Wiley 16.11.2018; Wiley Subscription Services, Inc; John Wiley and Sons Inc
• Subjects: Biocatalysis; Biochemistry & Molecular Biology; Chemical reactions; Chemistry, Medicinal; Computer applications; Crystal structure; Crystallography, X-Ray; C−C coupling; Destabilization; electron transfer; enzyme catalysis; Escherichia coli - genetics; Intermediates; Kinetics; Life Sciences & Biomedicine; Magnetic resonance spectroscopy; Models, Molecular; NMR spectroscopy; Oxidation-Reduction; Pharmacology & Pharmacy; Pichia - enzymology; radical reactions; reaction mechanisms; Science & Technology; Transketolase; Transketolase - chemistry; Yeast; C-C coupling; CARBENE; COMPLEX; INTERPLAY; DESIGN; DIPHOSPHATE-DEPENDENT ENZYMES; radical reactions; reaction mechanisms; REACTIVITY; enzyme catalysis; PICHIA-STIPITIS; DISTORTIONS; electron transfer; THIAMIN DIPHOSPHATE; INTERMEDIATE; C−C coupling
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.201800378

Transketolase (TK) catalyzes a reversible transfer of a two‐carbon (C2) unit between phosphoketose donors and phosphoaldose acceptors, for which the group‐transfer reaction that follows a one‐ or two‐electron mechanism and the force that breaks the C2“−C3” bond of the ketose donors remain unresolved. Herein, we report ultrahigh‐resolution crystal structures of a TK (TKps) from Pichia stipitis in previously undiscovered intermediate states and support a diradical mechanism for a reversible group‐transfer reaction. In conjunction with MS, NMR spectroscopy, EPR and computational analyses, it is concluded that the enzyme‐catalyzed non‐Kekulé diradical cofactor brings about the C2“−C3” bond cleavage/formation for the C2‐unit transfer reaction, for which suppression of activation energy and activation and destabilization of enzymatic intermediates are facilitated. Moving C2 units: Transketolase from P. stipitis catalyzes a reversible C2‐transfer radical mechanism, in which the thiamine diphosphate cofactor is subject to enzyme‐catalyzed isomerization to form non‐Kekulé diradicals required for bond cleavage/formation because of suppression of the activation energy and activation and destabilization of intermediates.