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

doi:10.13560/j.cnki.biotech.bull.1985.2021-0274

摘要/Abstract

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

孟晓建, 于建东, 郑小梅, 郑平, 李志敏, 孙际宾, 叶勤. 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[J]. 生物技术通报, 2021, 37(12): 180-190.

MENG Xiao-jian, YU Jian-dong, ZHENG Xiao-mei, ZHENG Ping, LI Zhi-min, SUN Ji-bin, YE Qin. Regulations of Small-molecules Metabolites on Hexokinase and Pyruvate Kinase in Aspergillus niger[J]. Biotechnology Bulletin, 2021, 37(12): 180-190.

图/表 9

表1 本研究所用的菌株、质粒与引物

Table 1 Strains,plasmids and primers used in this study

Primer name	Primer sequence（5'-3'）	Source
HxkA-F	gcggcggtggctcctctagaATGGTTGG- AATCGGTCCTAAG	This study
HxkA-R	ccgtcgcggtcgactctagaTTATAGCA- GGGTCTTCATGTC	This study
PkiA-F	gcggcggtggctcctctagaATGGCCGC- CAGCTCTTCCC	This study
PkiA-R	ccgtcgcggtcgactctagaTTACTCAGC- CAGGCCAAG	This study
GV-F	CGGAGATTCGTCGCCTAATGTC	This study
GV-R	CCGTCGGTCGCAATACAATCAC	This study

图1 己糖激酶与丙酮酸激酶黑曲霉重组表达菌株构建的PCR扩增电泳图 A:己糖激酶HxkA基因与丙酮酸激酶PkiA基因DNA序列的PCR扩增电泳图,M:Trans 8K DNA marker;1:HxkA基因;2:PkiA基因;B:黑曲霉重组表达菌株AnG1-HxkA与AnG1-PkiA的基因组PCR验证电泳图,M:Trans 8K DNA marker;NC:阴性对照;1-5:AnG1-HxkA各转化子的基因组PCR验证的电泳图,6-10:AnG1-PkiA各转化子的基因组PCR验证的电泳图

Fig. 1 Electrophoretogram of PCR products used for the construction of hexokinase（HxkA）and pyruvate kinase（PkiA）expressed A. niger strains A:PCR products of DNA sequences of hexokinase gene HxkA and pyruvate kinase gene PkiA. M:Trans 8K DNA marker. 1:HxkA gene. 2:PkiA gene. B:Genome PCR verification of recombinant expressing A. niger strain AnG1-HxkA and AnG1-PkiA. M:Trans 8K DNA marker. NC:Negative control. 1-5:The genome PCR verification of the transformants of AnG1-HxkA. 6-10:The genome PCR verification of the transformants of AnG1-PkiA

图2 己糖激酶与丙酮酸激酶GST融合蛋白亲和层析纯化前后的SDS-PAGE电泳图 A:GST-HxkA蛋白亲和层析前后的 SDS-PAGE电泳图,M:蛋白质分子量标准;1:GST-HxkA亲和层析前的SDS-PAGE电泳图;2:GST-HxkA经亲和层析后的SDS-PAGE电泳图;B:GST-PkiA蛋白亲和层析前后的 SDS-PAGE电泳图,M:蛋白质分子量标准;1:GST-PkiA亲和层析前的SDS-PAGE电泳图;2:GST-PkiA经亲和层析后的SDS-PAGE电泳图

Fig.2 SDS-PAGE of the recombinant GST-fused HxkA and PkiA proteins before and after protein purification by affinity chromatography A:SDS-PAGE of GST-HxkA before and after affinity chromatography. M:Protein marker. 1:SDS-PAGE of GST-HxkA before affinity chromatography. 2:SDS-PAGE of recombinant GST-HxkA after affinity chromatography. B:SDS-PAGE of GST-PkiA before and after affinity chromatography. M:Protein marker. 1:SDS-PAGE of GST-PkiA before affinity chromatography. 2:SDS-PAGE of GST-PkiA after affinity chromatography

图3 小分子代谢物对黑曲霉己糖激酶（A）与丙酮酸激酶酶活（B）影响的检测结果

Fig. 3 Effects of metabolites on the activities of HxkA（A）and PkiA（B）in A. niger * P<0.05,**P<0.01

图4 不同浓度的关键小分子代谢物对黑曲霉己糖激酶（A）与丙酮酸激酶酶活（B）影响的检测结果虚线代表不同浓度的具有激活作用的小分子代谢物存在时酶活变化;实线代表不同浓度的具有抑制作用的小分子代谢物存在时酶活变化

Fig.4 Effects of key metabolites under different concent-rations on the activities of HxkA（A）and PkiA（B）in A. niger The dotted lines refer to changes in enzyme activity in the presence of small molecule metabolites with activation at different concentrations. Solid lines refer to changes in enzyme activity in the presence of small molecule metabolites with inhibition at different concentrations

图5 黑曲霉己糖激酶与丙酮酸激酶的多序列比对结果 A:黑曲霉己糖激酶与不同来源己糖激酶的多序列比对结果,红色五角星为己糖激酶的底物结合位点;B:黑曲霉丙酮酸激酶与不同来源丙酮酸激酶的多序列比对结果,红色五角星为丙酮酸激酶的活性位点

Fig.5 Multiple sequence alignment of HxkA and PkiA from A. niger A:Multiple sequence alignment of HxkA from A. niger with hexokinases from different species,the substrate binding sites of hexokinases are labeled with red stars.B:Multiple sequence alignment of PkiA from A. niger with pyruvate kinases from different species,the catalytic active sites of pyruvate kinases are labeled with red stars

图6 基于同源建模的黑曲霉己糖激酶（A）与丙酮酸激酶（B）三维结构模拟结果 A:蓝色与绿色分别为黑曲霉己糖激酶的2个亚基,红色为黑曲霉己糖激酶的活性位点;B:蓝色、绿色、紫色与黄色分别为黑曲霉丙酮酸激酶的4个亚基,红色为黑曲霉丙酮酸激酶的活性位点

Fig.6 Simulated 3D structure results of HxkA（A）and PkiA（B）from A. niger based on homologous modeling A:Protein structures of 2 sub-units of HxkA in A. niger shown in blue and green;the catalytic active sites of HxkA were labeled in red. B:Protein structures of 4 sub-units of PkiA shown in blue,green,purple and yellow;the catalytic active sites of PkiA are labeled in red

图7 己糖激酶HxkA与关键小分子代谢物的分子对接模拟结果 A:黑曲霉己糖激酶与6-磷酸果糖的分子对接模拟结果;B:己糖激酶与PEP的分子对接模拟结果;C:己糖激酶与ADP的分子对接模拟结果;D:己糖激酶与ATP的分子对接模拟结果。黄色虚线表示氨基酸与关键小分子代谢物形成的氢键,红色数字表示键长

Fig.7 Molecular docking simulation of HxkA with key metabolites A-D:Molecular docking simulation of HxkA with fructose 6-phosphate,PEP,ADP,and ATP,respectively. The hydrogen bonds between the amino acids and key metabolites are represented as yellow dash lines,the hydrogen bond distances are represented in red numbers

图8 丙酮酸激酶PkiA与关键小分子代谢物的分子对接模拟结果 A:黑曲霉丙酮酸激酶与ADP的分子对接模拟结果;B:丙酮酸激酶与1,6-二磷酸果糖的分子对接模拟结果;C:丙酮酸激酶与苹果酸的分子对接模拟结果;D:丙酮酸激酶与富马酸的分子对接模拟结果。黄色虚线表示氨基酸与关键小分子代谢物形成的氢键,红色数字表示键长。

Fig.8 Molecular docking simulation of PkiA and key metabolites A-D:Molecular docking simulation of PkiA with ADP,fructose 1,6-diphosphate,malic acid,and fumaric acid,respectively. The hydrogen bonds between the amino acids and key metabolites are represented as yellow dash lines;the hydrogen bond distances are represented in red numbers.

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