不同抑制剂对谷氨酸棒状杆菌磷酸烯醇式丙酮酸羧化酶酶活的影响

生物技术通报 ›› 2013, Vol. 0 ›› Issue (7): 172-178.

不同抑制剂对谷氨酸棒状杆菌磷酸烯醇式丙酮酸羧化酶酶活的影响

吴新阳^1，3 裴广胜^1，3 郑小梅^2，3 曹国强^2，3 刘娇^2，3 郑平^2，3 王敏¹ 孙际宾^2，3

（1.天津科技大学生物工程学院，天津 300457；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；）

收稿日期:2013-01-04 修回日期:2013-07-19 出版日期:2013-07-19 发布日期:2013-09-02
作者简介:吴新阳，男，硕士研究生，研究方向：生物化学与分子生物学；E-mail：sanwuxinyang@163.com；裴广胜为并列第一作者
基金资助:
国家重点基础研究发展计划（973 计划）（2009CB724703），中科院知识创新方向性项目（KSCX2-EW-Q-13），天津市应用基础与前沿技术研究计划重点项目（10JCZDJC16200，11ZCZDSY08400，11ZCZDSY08500）

Phosphoenolpyruvate Carboxylase from Corynebacterium glutamicum Inhibited by Diverse Biochemicals

Wu Xinyang^1，3 Pei Guangsheng^1，3 Zheng Xiaomei^2，3 Cao Guoqiang^2，3 Liu Jiao^2，3 Zheng Ping^2，3 Wang Min¹ Sun Jibin^2，3

（1. School of Biological Engineering，Tianjin University of Science and Technology，Tianjin 300457；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）

Received:2013-01-04 Revised:2013-07-19 Published:2013-07-19 Online:2013-09-02

摘要/Abstract

摘要： 磷酸烯醇式丙酮酸羧化酶（Phosphoenolpyruvate carboxylase，PPC）是生物体碳代谢四碳回补途径的重要酶，是很多生物基化学品合成途径中的关键反应。为揭示谷氨酸棒状杆菌的PPC酶受体内代谢物影响的情况，对谷氨酸棒状杆菌Corynebacterium glutamicum ATCC13032的ppc基因进行了PCR克隆，成功地在大肠杆菌中诱导表达为可溶性蛋白。利用Ni柱亲和层析纯化了该蛋白，酶活性测定表明其具有磷酸烯醇式丙酮酸羧化酶活性。细致研究了各种抑制剂，如柠檬酸、异柠檬酸、富马酸、琥珀酸、苹果酸、谷氨酸、天冬氨酸等对PPC酶活的影响，证实天冬氨酸和苹果酸是谷氨酸棒杆菌PPC的强抑制剂，并首次发现PPC也受到N-乙酰谷氨酰胺的抑制作用。

关键词: 磷酸烯醇式丙酮酸羧化酶, 谷氨酸棒状杆菌, 抑制剂, N-乙酰谷氨酰胺

Abstract: Phosphoenolpyruvate carboxylase（PPC）, an enzyme involved in replenishment of the four-carbon dicarboxylic acid intermediate pool of the TCA cycle, plays an important role in both biological and biotechnological synthesis pathways. To discover the effect of diverse inhibitors on the activity of PPC, the full-length sequence of the ppc gene from Corynebacterium glutamicum ATCC13032 was cloned and expressed as soluble form in Escherichia coli. The recombinant PPC was purified with Ni-Column. The purified enzyme showed high phosphoenolpyruvate carboxylase activity, and was inhibited by diverse inhibitors such as citrate, isocitrate, fumarate, glutamate, aspartate, malate etc. Aspartate and malate were the strongest inhibitors. For the first time, the inhibition of PPC activity by N-acetyl glutamine was clearly demonstrated in a glutamate-producing strain of C. glutamicum.

Key words: Phosphoenolpyruvate carboxylase, Corynebacterium glutamicum, Inhibitor, N-acetyl glutamine.

吴新阳,裴广胜,郑小梅,曹国强,刘娇,郑平,王敏,孙际宾. 不同抑制剂对谷氨酸棒状杆菌磷酸烯醇式丙酮酸羧化酶酶活的影响[J]. 生物技术通报, 2013, 0(7): 172-178.

Wu Xinyang,Pei Guangsheng,Zheng Xiaomei,Cao Guoqiang,Liu Jiao,Zheng Ping,Wang Min,Sun Jibin. Phosphoenolpyruvate Carboxylase from Corynebacterium glutamicum Inhibited by Diverse Biochemicals[J]. Biotechnology Bulletin, 2013, 0(7): 172-178.

参考文献

[1] Nui M, Dumay V, Zahn K, et al. Structural and functional analysis of the phosphoenolpyruvate carboxylase gene from the purple nonsulfur bacterium Rhodopseudomonas palustris No 7[J]. J Bacteriol, 1997, 179（15）：4942-4945.
[2] Eikmanns BJ, Follettie MT, Griot MU, et al. The phosphoenolpyruvate carboxylase gene of Corynebacterium glutamicum：molecular cloning, nucleotide sequence, and expression[J]. Molecular & General Genetics, 1989, 218（2）：330-339.
[3] Izui K, Taguchi M, Morikawa M, et al. Regulation of Escherichia coli phosphoenolpyruvate carboxylase by multiple effectors in vivo. II. Kinetic studies with a reaction system containing physiological concentrations of ligands[J]. J Biochem, 1981, 90（5）：1321-1331.
[4] Schwitzguebel JP, Ettlinger L. Phosphoenolpyruvate carboxylase from Acetobacter aceti[J]. Arch Microbiol, 1979, 122（1）：109-115.
[5] Mori M, Shiio I. Purification and some properties of phosphoenolpyr-uvate carboxylase from Brevibacterium flavum and its aspartate-overproducing mutant[J]. J Biochem, 1985, 97：1119-1128.
[6] Mori M, Shiio I. Multiple interaction of fructose 1, 6 biphosphate and other effectors on phosphoenolpyruvate carboxylase from Brevibacte-rium flavum and its aspartate-producing mutant[J]. Agric Biol Chem, 1986, 50：2605-2614.
[7] Mori M, Shiio I. Synergistic inhibition of phosphoenolpyruvate carboxylase by aspartate and 2-oxoglutarate in Brevibacterium flavum[J]. J Biochem, 1985, 98：1621-1630.
[8] Ozaki H, Shiio I. Regulation of the TCA and glyoxylate cycles in Bre-vibacterium flavum：regulation of phosphoenolpyruvate carboxylase and pyruvate kinase[J]. J Biochem, 1969, 66：297-311.
[9] O’Regan M, Thierbach G, Bachmann B, et al. Cloning and nucleotide sequence of the phosphoenolpyruvate carboxylase-coding gene of Corynebacterium glutamicum ATCC13032[J]. Gene, 1989, 77（2）：237-51.
[10] Delaunay S, Lapujade PD, Engasser JM, et al. Glutamate as an inhibitor of phosphoenolpyruvate carboxylase activity in Corynebacterium glutamicum[J]. J Ind Microbiol Biotechnol, 2004, 31（4）：183-188.
[11] Sambrook J, David W. Molecular cloning：a laboratory manual[M].3rd ed. Cold Spring Harbor Laboratory Press, 2002：99-102.
[12] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72：248-254.
[13] Izui K. Kinetic studies on the allosteric nature of phosphoenolpyru-vate carboxylase from Escherichia coli[J]. J Biochem, 1970, 68：227-238.
[14] Wohl RC, Markus G. Phosphoenolpyruvate carboxylase of Escheri-chia coli. Purification and some properties[J]. J Biol Chem, 1972, 247（18）：5785-5792.
[15] Takai K, Sako Y, Uchida A. ppc, the gene for phosphoenolpyruvate carboxylase from an extremely thermophilic bacterium, Rhodother-mus obamensis：cloning, sequencing and overexpression in Esche-richia coli[J]. Microbiology, 1998, 144：1423-1434.
[16] Schuller KA, Turpin DH, Plaxton WC. Metabolite regulation of partially purified soybean nodule phosphoenolpyruvate carboxyl-ase[J]. Plant Physiol, 1990, 94（3）：1429-1435.
[17] Chen LM, Omiya T, Hata S, et al. Molecular characterization of a ph-osphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties[J]. Plant and Cell Physiology, 2002, 43（2）：159-169.
[18] Toru Nakanishi. Roles of Ammonium and chloride ions in the conv-ersion of L-glutamic acid fermentation to L-glutamine and N-acetyl-L-glutamine fermentation by corynebacterium glutamicum[J]. Journal of Fermentation Technology, 1978, 56（3）：179-188.
[19] 赵艳, 陈丽梅, 李昆志.磷酸烯醇式丙酮酸羧化酶的分子结构研究进展[J].生物技术通报, 2007（5）：9-13.