Change of Nucleotide Specificity and Enhancement of Catalytic Efficiency in Single Point Mutants of Vibrio harveyi Aldehyde Dehydrogenase

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• 作者：Lei Zhang ; Bijan Ahvazi ; Rose Szittner ; Alice Vrielink ; and Edward Meighen
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：August 31, 1999
• 年：1999
• 卷：38
• 期：35
• 页码：11440 - 11447
• 全文大小：108K
• 年卷期：v.38,no.35(August 31, 1999)
• ISSN：1520-4995

文摘

The fatty aldehyde dehydrogenase from the luminescent bacterium, Vibrio harveyi (Vh-ALDH),is unique with respect to its high specificity for NADP⁺ over NAD⁺. By mutation of a single threonineresidue (Thr175) immediately downstream of the _B strand in the Rossmann fold, the nucleotide specificityof Vh-ALDH has been changed from NADP⁺ to NAD⁺. Replacement of Thr175 by a negatively chargedresidue (Asp or Glu) resulted in an increase in k_cat/K_m for NAD⁺ relative to that for NADP⁺ of up to5000-fold due to a decrease for NAD⁺ and an increase for NADP⁺ in their respective Michaelis constants(K_a). Differential protection by NAD⁺ and NADP⁺ against thermal inactivation and comparison of thedissociation constants of NMN, 2'-AMP, 2'5'-ADP, and 5'-AMP for these mutants and the wild-typeenzyme clearly support the change in nucleotide specificity. Moreover, replacement of Thr175 with polarresidues (N, S, or Q) demonstrated that a more efficient NAD⁺-dependent enzyme T175Q could be createdwithout loss of NADP⁺-dependent activity. Analysis of the three-dimensional structure of Vh-ALDHwith bound NADP⁺ showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphateof NADP⁺. Replacement with glutamic acid or glutamine strengthened interactions with NAD⁺ andindicated why threonine would be the preferred polar residue at the nucleotide recognition site in NADP⁺-specific aldehyde dehydrogenases. These results have shown that the size and the structure of the residueat the nucleotide recognition site play the key roles in differentiating between NAD⁺ and NADP⁺interactions while the presence of a negative charge is responsible for the decrease in interactions withNADP⁺ in Vh-ALDH.