Nara Prefecture Institute of Industrial Development, Nara, Nara, Japan.
Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technologygrid.260493.a, Ikoma, Nara, Japan.
Microbiol Spectr. 2022 Jun 29;10(3):e0082222. doi: 10.1128/spectrum.00822-22. Epub 2022 May 11.
In the yeast Saccharomyces cerevisiae, -acetyl glutamate kinase (NAGK), which catalyzes the phosphorylation of -acetyl glutamate to form -acetyl glutamyl-5-phosphate, is one of the rate-limiting enzymes in the ornithine and arginine biosynthetic pathways. NAGK activity is strictly regulated via feedback inhibition by the end product, arginine. We previously reported that the Thr340Ile variant of NAGK was insensitive to arginine feedback inhibition and that the interaction between Lys336 and Thr340 in NAGK may be important for arginine recognition. In the present study, we demonstrated that amino acid changes of Thr340 to Ala, Leu, Arg, Glu, Ile, and Asn removed arginine feedback inhibition, although the Thr340Ser variant was subject to the feedback inhibition. Therefore, these results indicate that the arginine-binding cavity formed via the interaction between the carbonyl group in the main chain of Lys336 and the hydroxyl group in the side chain of the residue at position 340 is critical for arginine recognition of NAGK. In addition, we newly identified two mutations in the gene encoding the Cys119Tyr or Val267Ala variant of NAGK of sake yeast mutants with intracellular ornithine accumulation. Although it is unlikely that Cys119 and Val267 are directly involved in arginine recognition, we found here that two variants of NAGK were insensitive to arginine feedback inhibition and contributed to high-level production of ornithine. Structural analysis of NAGK suggests that these two amino acid substitutions influence the sensitivity to Arg feedback inhibition through alterations in local conformation around each residue. Ornithine has a number of physiological benefits in humans. Thus, an Orn-rich alcoholic beverage is expected to relieve feelings of fatigue after drinking. In the yeast Saccharomyces cerevisiae, -acetyl glutamate kinase (NAGK) encoded by the gene catalyzes the second step in ornithine and arginine biosynthesis, and its activity is subjected to feedback inhibition by arginine. Here, we revealed a role of key residues in the formation of the arginine-binding cavity which is critical for arginine recognition of NAGK. In addition, we analyzed novel arginine feedback inhibition-insensitive variants of NAGK in sake yeast mutants with ornithine overproduction and proposed that the amino acid substitutions in the NAGK variants destabilize the arginine-binding cavity, leading to the lower sensitivity to arginine feedback inhibition of NAGK activity. These findings provide new insight into the allosteric regulation of NAGK activity and will help to construct superior industrial yeast strains for high-level production of ornithine.
在酵母酿酒酵母中，-乙酰谷氨酸激酶（NAGK）催化-乙酰谷氨酸磷酸化形成-乙酰谷氨酸-5-磷酸，是鸟氨酸和精氨酸生物合成途径中的限速酶之一。NAGK 活性通过终产物精氨酸的反馈抑制严格调节。我们之前报道过，NAGK 的 Thr340Ile 变体对精氨酸反馈抑制不敏感，并且 NAGK 中 Lys336 和 Thr340 之间的相互作用可能对精氨酸识别很重要。在本研究中，我们证明了 Thr340 突变为 Ala、Leu、Arg、Glu、Ile 和 Asn 可消除精氨酸反馈抑制，尽管 Thr340Ser 变体仍受反馈抑制。因此，这些结果表明，通过 Lys336 主链上的羰基和残基 340 侧链上的羟基之间的相互作用形成的精氨酸结合腔对于 NAGK 的精氨酸识别至关重要。此外，我们在具有细胞内鸟氨酸积累的清酒酵母突变体的编码 NAGK 的 Cys119Tyr 或 Val267Ala 变体的 基因中发现了两个新突变。尽管不太可能 Cys119 和 Val267 直接参与精氨酸识别，但我们在这里发现两种 NAGK 变体对精氨酸反馈抑制不敏感，并有助于高水平生产鸟氨酸。NAGK 的结构分析表明，这两个氨基酸取代通过改变每个残基周围的局部构象来影响对 Arg 反馈抑制的敏感性。鸟氨酸在人体中有许多生理益处。因此，富含鸟氨酸的酒精饮料有望缓解饮酒后的疲劳感。在酵母酿酒酵母中，由 基因编码的 -乙酰谷氨酸激酶（NAGK）催化鸟氨酸和精氨酸生物合成的第二步，其活性受精氨酸的反馈抑制。在这里，我们揭示了关键残基在精氨酸结合腔形成中的作用，该结合腔对于 NAGK 的精氨酸识别至关重要。此外，我们分析了清酒酵母突变体中鸟氨酸过量产生的新型精氨酸反馈抑制不敏感的 NAGK 变体，并提出 NAGK 变体中的氨基酸取代使精氨酸结合腔不稳定，导致 NAGK 活性对精氨酸反馈抑制的敏感性降低。这些发现为 NAGK 活性的变构调节提供了新的见解，并有助于构建用于高水平生产鸟氨酸的优良工业酵母菌株。
