Alkylpyrazines are important contributors to the flavor of traditional fermented foods. Here, we studied the synthetic mechanisms of 2,5-dimethylpyrazine (2,5-DMP) and 2,3,5-trimethylpyrazine (TMP). Substrate addition, whole-cell catalysis, stable isotope tracing experiments and gene manipulation revealed that l-threonine is the starting point involving l-threonine-3-dehydrogenase (TDH) and three uncatalyzed reactions to form 2,5-DMP. TDH catalyzes the oxidation of l-threonine. The product of this reaction is l-2-amino-acetoacetate which is known to be unstable and can decarboxylate to form aminoacetone. It is proposed that aminoacetone spontaneously converts to 2,5-DMP in a pH-dependent reaction, via 3,6-dihydro-2,5-DMP. 2-Amino-3-ketobutyrate CoA ligase (KBL) catalyzes the cleavage of l-2-amino-acetoacetate, the product of TDH, into glycine and acetyl-CoA in the presence of CoA. Inactivation of KBL could improve the production of 2,5-DMP. Besides 2,5-DMP, 2,3,5-trimethylpyrazine (TMP) can also be generated by B. subtilis 168 by using l-threonine and d-glucose as substrates and TDH as the catalytic enzyme.

Importance Despite the flavor contribution and commercial value, the synthetic mechanisms of alkylpyrazines by microorganisms remain poorly understood. This study revealed the substrate, intermediates and related enzymes for the synthesis of 2,5-dimethylpyrazine (2,5-DMP), which differs from the previous reports about the synthesis of 2,3,5,6-tetramethylpyrazine (TTMP). The synthetic mechanism described here can also explain the production of 2,3,5-trimethylpyrazine (TMP). The results provide insights into an alkylpyrazine's synthetic pathway involving l-threonine-3-dehydrogenase as the catalytic enzyme and l-threonine as the substrate.

烷基吡嗪是传统发酵食品风味的重要贡献者。在这里，我们研究了2,5-二甲基吡嗪（2,5-DMP）和2,3,5-三甲基吡嗪（TMP）的合成机理。底物添加，全细胞催化，稳定的同位素示踪实验和基因操作表明，升-苏氨酸是涉及的起点 升-苏氨酸-3-脱氢酶（TDH）和三个未催化的反应形成2,5-DMP。TDH催化氧化升-苏氨酸。该反应的产物是1-2-氨基-乙酰乙酸酯，其已知是不稳定的并且可以脱羧形成氨基丙酮。提出氨基丙酮在pH依赖性反应中通过3，6-二氢-2，5-DMP自发地转化为2，5-DMP。在CoA存在下，2-氨基-3-酮丁酸酯CoA连接酶（KBL）催化TDH的1-2-氨基-乙酰乙酸酯裂解为甘氨酸和乙酰CoA。KBL的失活可以提高2，5-DMP的产生。除了2,5-DMP之外，枯草芽孢杆菌168还可以使用以下方法生成2,3,5-三甲基吡嗪（TMP）升-苏氨酸和 d-葡萄糖作为底物，TDH作为催化酶。

重要性尽管有风味贡献和商业价值，但微生物对烷基吡嗪的合成机理仍知之甚少。这项研究揭示了合成2,5-二甲基吡嗪（2,5-DMP）的底物，中间体和相关酶，这与以前有关2,3,5,6-四甲基吡嗪（TTMP）合成的报道不同。此处描述的合成机理也可以解释2,3,5-三甲基吡嗪（TMP）的产生。结果为深入了解烷基吡嗪的合成途径提供了见解。升-苏氨酸-3-脱氢酶作为催化酶和 升-苏氨酸为底物。