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J. Biol. Chem., Vol. 279, Issue 32, 33946-33957, August 6, 2004

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Structures of Human Cytosolic NADP-dependent Isocitrate Dehydrogenase Reveal a Novel Self-regulatory Mechanism of Activity^*

Xiang Xu, Jingyue Zhao, Zhen Xu, Baozhen Peng, Qiuhua Huang, Eddy Arnold¶, and Jianping Ding¶||

From the Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China, the State Key Laboratory for Medical Genomics, Shanghai Institute of Hematology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China, and the ^¶Center for Advanced Biotechnology and Medicine and Rutgers University Department of Chemistry and Chemical Biology, Piscataway, New Jersey 08854-5638

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to -ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca²⁺ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved -helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled -helix in the semi-open, intermediate form; and an -helix in the closed, active form. The side chain of Asp²⁷⁹ of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser⁹⁴ (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

Received for publication, April 19, 2004 , and in revised form, May 24, 2004.

The atomic coordinates and structure factors (codes 1T09 and 1T0L) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Natural Science Foundation of China Grants 30125011, 30170223, and 30130080; 863 Hi-Tech Program Grants 2001AA235071, 2001AA233021, and 2002BA711A13; and Chinese Academy of Sciences Grant KSCX1-SW-17 (to J. D.). This work was also supported in part by National Institutes of Health Grants AI 27690 and GM 66671 (to E. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Rd., Shanghai 200031, China. Tel.: 86-21-54921619; Fax: 86-21-54921116; E-mail: jpding{at}sibs.ac.cn.

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