Significance of Asn-77 and Trp-78 in the Catalytic Function of Undecaprenyl Diphosphate Synthase of Micrococcus luteus B-P 26.
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概要
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The primary structures of cis-prenyltransferases are completely different from those of trans-prenyltransferases. To obtain information about amino acid residues relating to catalytic function, random mutation of the undecaprenyl diphosphate synthase gene of Micrococcus luteus B-P 26 was carried out to construct a mutated gene library using an error-prone polymerase chain reaction. From the library, the mutants showing poor enzymatic activity were selected by the colony autoradiography method. Among 31 negative clones selected from 3, 000 mutants, two clones were found to contain only one amino acid substitution at either Asn-77 or Trp-78. To determine the functional roles of these interesting residues, we prepared six mutated enzymes with substitutions at residues Asn-77 or Trp-78 by site-directed mutagenesis. Substitution of Asn-77 with Ala, Asp, or Gln resulted in a dramatic decrease in catalytic activity. but the Km values for both aBylic and homoallylic substrates of these mutant enzymes were comparable to those of the wild-type. On the other hand, three Trp-78 mutants, W781, W78R, and W78D, showed 5-20-fold increased Km values for farnesyl diphosphate but not for Z-geranylgeranyl diphosphate. However, these mutants showed moderate levels of enzymatic activity and comparable Km values for isopentenyl diphosphate to that of the wild-type. These results suggest that the Asn-Trp motif is involved in the binding of farnesyl diphosphate and enzymatic catalysis.
- 社団法人 日本生化学会の論文
著者
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Koyama Tanetoshi
Institute For Chemical Reaction Science Tohoku University
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Nishino Tokuzo
Department Of Biochemistry And Engineering Faculty Of Engineering Tohoku University
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ZHANG Yuan-Wei
Institute for Chemical Reaction Science, Tohoku University
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Yoshizaki Hideki
Department Of Biochemistry And Engineering Tohoku University
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Fujikura Keitaro
Institute For Chemical Reaction Science Tohoku University
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