Chemoenzymatic synthesis of 3-ethyl-2,5-dimethylpyrazine by L-threonine 3-dehydrogenase and 2-amino-3-ketobutyrate CoA ligase/L-threonine aldolase

Tomoharu Motoyama,Noriyuki Miyoshi,Shogo Nakano,Fumihito Hasebe,Sohei Ito,Ryo Miyata,Shigenori Kumazawa

doi:10.1038/s42004-021-00545-8

Tomoharu Motoyama, Noriyuki Miyoshi + Show 5 more

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https://doi.org/10.1038/s42004-021-00545-8

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Abstract

Pyrazines are typically formed from amino acids and sugars in chemical reactions such as the Maillard reaction. In this study, we demonstrate that 3-ethyl-2,5-dimethylpyrazine can be produced from L-Thr by a simple bacterial operon. We conclude that EDMP is synthesized chemoenzymatically from L-Thr via the condensation reaction of two molecules of aminoacetone and one molecule of acetaldehyde. Aminoacetone is supplied by L-threonine 3-dehydrogenase using L-Thr as a substrate via 2-amino-3-ketobutyrate. Acetaldehyde is supplied by 2-amino-3-ketobutyrate CoA ligase bearing threonine aldolase activity from L-Thr when CoA was at low concentrations. Considering the rate of EDMP production, the reaction intermediate is stable for a certain time, and moderate reaction temperature is important for the synthesis of EDMP. When the precursor was supplied from L-Thr by these enzymes, the yield of EDMP was increased up to 20.2%. Furthermore, we demonstrate that this reaction is useful for synthesizing various alkylpyrazines.

Highlights

Pyrazines are typically formed from amino acids and sugars in chemical reactions such as the Maillard reaction
This group reported that aminoacetone, which is produced by L-threonine 3-dehydrogenase (TDH) could be used as a component in the synthesis of DPO14; DPO was synthesized through the condensation and cyclization of aminoacetone and N-alanyl-aminoacetone using many unassigned enzymes[15]
We show that EDMP can be synthesized from only LThr by combinational usage of TDH and ketobutyrate CoA ligase (KBL)

Summary

Introduction

Pyrazines are typically formed from amino acids and sugars in chemical reactions such as the Maillard reaction. Aloisine A is utilized as a therapy for Alzheimer’s disease[6], pyrazinamide is used as an anti-tuberculosis drug[7], and favipiravir is used as an anti-RNA antiviral drug targeting influenza and coronavirus[8] Based on these considerations, it is no doubt that pyrazines are important compounds for both basic and applied studies. Papenfort et al reported that 3,5-dimethylpyrazine-2-ol (DPO), which controls biofilm formation, was produced by metabolizing L-Thr in Vibrio cholerae This group reported that aminoacetone, which is produced by L-threonine 3-dehydrogenase (TDH) could be used as a component in the synthesis of DPO14; DPO was synthesized through the condensation and cyclization of aminoacetone and N-alanyl-aminoacetone using many unassigned enzymes[15]. We hypothesize that some alkylpyrazines can be produced under normal conditions (e.g., normal temperature and water systems) by combinations of enzymatic and chemical reactions

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