生物技术通报 ›› 2021, Vol. 37 ›› Issue (12): 180-190.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0274

• 研究报告 • 上一篇    下一篇

小分子代谢物对黑曲霉己糖激酶和丙酮酸激酶的酶活调控

孟晓建1,2,3(), 于建东2,3, 郑小梅2,3,4(), 郑平2,3,4, 李志敏1(), 孙际宾2,3,4, 叶勤1   

  1. 1.华东理工大学生物反应器工程国家重点实验室,上海 200237
    2.中国科学院系统微生物工程重点实验室,天津 300308
    3.中国科学院天津工业生物技术研究所,天津 300308
    4.国家合成生物技术创新中心,天津 300308
  • 收稿日期:2021-03-10 出版日期:2021-12-26 发布日期:2022-01-19
  • 作者简介:孟晓建,男,硕士研究生,研究方向:酶的代谢调控;E-mail: mengxj@tib.cas.cn
  • 基金资助:
    国家自然科学基金项目(31370113);国家自然科学基金项目(32070082);天津市合成生物技术创新能力提升行动项目(TSBICIP-PTJS-003)

Regulations of Small-molecules Metabolites on Hexokinase and Pyruvate Kinase in Aspergillus niger

MENG Xiao-jian1,2,3(), YU Jian-dong2,3, ZHENG Xiao-mei2,3,4(), ZHENG Ping2,3,4, LI Zhi-min1(), SUN Ji-bin2,3,4, YE Qin1   

  1. 1. State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237
    2. Key Laboratory of Systems Microbial Biotechnology,Chinese Academy of Sciences,Tianjin 300308
    3. Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences,Tianjin 300308
    4. National Technology Innovation Center of Synthetic Biology,Tianjin 300308
  • Received:2021-03-10 Published:2021-12-26 Online:2022-01-19

摘要:

黑曲霉是柠檬酸的工业生产菌株,糖酵解中的3个不可逆反应是柠檬酸积累的重要调控节点,但己糖激酶和丙酮酸激酶的代谢调控研究相对较少,本研究旨在加深对己糖激酶和丙酮酸激酶代谢调控的认识。首先,将黑曲霉己糖激酶和丙酮酸激酶进行内源过表达,经GST亲和层析纯化后重组酶的比酶活分别为(4.86±0.14)U/mg和(1.83±0.02)U/mg。通过小分子代谢物对其酶活影响的检测,发现己糖激酶受果糖-6-磷酸、磷酸烯醇式丙酮酸、ADP和ATP的抑制,而丙酮酸激酶被果糖-1,6-二磷酸、苹果酸和富马酸抑制,同时存在底物ADP的前馈激活作用。进一步通过蛋白三维结构建模与蛋白-小分子代谢物对接模拟分析,发现黑曲霉己糖激酶底物结合位点Asn210及其附近的氨基酸是与小分子代谢物互作的关键位点,而丙酮酸激酶位于别构效应结构域的Thr416、Thr417与Trp470则为关键小分子代谢物的重要结合位点。本研究系统鉴定了小分子代谢物对己糖激酶与丙酮酸激酶的代谢调控关系并预测了其别构调控位点,加深了对黑曲霉糖酵解调控机制的认识。

关键词: 黑曲霉, 柠檬酸, 己糖激酶, 丙酮酸激酶, 代谢调控

Abstract:

Aspergillus niger is an industrial strain for citric acid production. Three irreversible reactions in the glycolysis pathway are important regulation nodes for citric acid accumulation. However,there are only few studies regarding the metabolic regulation of hexokinase and pyruvate kinase. This study aims to unveil the regulation of metabolic intermediates on hexokinase and pyruvate kinase in A. niger. First,the genes encoding hexokinase and pyruvate kinase were overexpressed in A. niger and the proteins were purified by GST affinity chromatography. The specific enzyme activities of the recombined hexokinase and pyruvate kinase were(4.86±0.14)U/mg and(1.83±0.02)U/mg,respectively. Then,the effects of metabolites on the activities of hexokinase and pyruvate kinase were measured. Fructose-6-phosphate,phosphoenolpyruvate,ADP and ATP were the inhibitors of hexokinase. As to pyruvate kinase,fructose-1,6-diphosphate,malic acid and fumaric acid presented inhibitory effects,while its substrate ADP showed feed-forward activation. Finally,the molecular docking of key metabolites with enzymes was simulated. It discovered that substrate binding site Asn210 of hexokinase and vital sites of its adjacent amino acids and key metabolites;while the Thr416,Thr417 and Trp470 of the pyruvate kinase in the allosteric domain were the vital binding sites for key metabolites. In this study,we systematically identified the metabolic regulation of metabolites on the hexokinase and pyruvate kinase and predicted their allosteric regulatory sites,which deepens the understanding of the regulatory mechanism of glycolysis in A. niger.

Key words: Aspergillus niger, citric acid, hexokinase, pyruvate kinase, metabolic regulation