Structural basis for the alteration of coenzyme specificity in a malate dehydrogenase mutant |
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Authors: | Tomita Takeo Fushinobu Shinya Kuzuyama Tomohisa Nishiyama Makoto |
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Affiliation: | Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Tokyo 113-8657, Japan. |
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Abstract: | To elucidate the structural basis for the alteration of coenzyme specificity from NADH toward NADPH in a malate dehydrogenase mutant EX7 from Thermus flavus, we determined the crystal structures at 2.0 A resolution of EX7 complexed with NADPH and NADH, respectively. In the EX7-NADPH complex, Ser42 and Ser45 form hydrogen bonds with the 2'-phosphate group of the adenine ribose of NADPH, although the adenine moiety is not seen in the electron density map. In contrast, although Ser42 and Ser45 occupy a similar position in the EX7-NADH complex structure, both the adenine and adenine ribose moieties of NADH are missing in the map. These results and kinetic analysis of site-directed mutant enzymes indicate (1) that the preference of EX7 for NADPH over NADH is ascribed to the recognition of the 2'-phosphate group by two Ser and Arg44, and (2) that the adenine moiety of NADPH is not recognized in this mutant. |
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Keywords: | Crystal structure Malate dehydrogenase Coenzyme specificity Thermostable enzyme |
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