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J. Biol. Chem., Vol. 280, Issue 17, 17068-17075, April 29, 2005

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Distinct Conformation-mediated Functions of an Active Site Loop in the Catalytic Reactions of NAD-dependent D-Lactate Dehydrogenase and Formate Dehydrogenase^*

Takeshi Shinoda, Kazuhito Arai, Mayu Shigematsu-Iida, Yoshirou Ishikura, Satoru Tanaka, Takashi Yamada, Matthew S. Kimber, Emil F. Pai¶, Shinya Fushinobu||, and Hayao Taguchi**

From the Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan, Affinium Pharmaceuticals Inc., Toronto, Ontario M5J 1V6, Canada, the ^¶Departments of Biochemistry and Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and ^||Department of Biotechnology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

The three-dimensional structures of NAD-dependent D-lactate dehydrogenase (D-LDH) and formate dehydrogenase (FDH), which resemble each other, imply that the two enzymes commonly employ certain main chain atoms, which are located on corresponding loop structures in the active sites of the two enzymes, for their respective catalytic functions. These active site loops adopt different conformations in the two enzymes, a difference likely attributable to hydrogen bonds with Asn⁹⁷ and Glu¹⁴¹, which are also located at equivalent positions in D-LDH and FDH, respectively. X-ray crystallography at 2.4-Å resolution revealed that replacement of Asn⁹⁷ with Asp did not markedly change the overall protein structure but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus D-LDH. The Asn⁹⁷ Asp mutant D-LDH exhibited virtually the same k_cat, but about 70-fold higher K_M value for pyruvate than the wild-type enzyme. For Paracoccus sp. 12-A FDH, in contrast, replacement of Glu¹⁴¹ with Gln and Asn induced only 5.5- and 4.3-fold increases in the K_M value, but 110 and 590-fold decreases in the k_cat values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu¹⁴¹ Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxy-acid dehydrogenase on the replacement of Glu¹⁴¹. These results indicate that the active site loops play different roles in the catalytic reactions of D-LDH and FDH, stabilization of substrate binding and promotion of hydrogen transfer, respectively, and that Asn⁹⁷ and Glu¹⁴¹, which stabilize suitable loop conformations, are essential elements for proper loop functioning.

Received for publication, January 26, 2005 , and in revised form, February 25, 2005.

^* This work was supported by a grant-in-aid for scientific research (to H. T.) from the Ministry of Education, Science, and Culture of Japan. 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. Tel.: 81-4-7124-1501 (ext. 3407); Fax: 81-4-7123-9767; E-mail: htaguchi{at}rs.noda.tus.ac.jp.

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