Mechanistic and Structural Analyses of the Roles of Arg409 and Asp402 in the Reaction of the Flavoprotein Nitroalkane Oxidase

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• 作者：Paul F. Fitzpatrick ; Dragana M. Bozinovski ; Annie H&eacute ; roux ; Patrick G. Shaw ; Michael P. Valley ; Allen M. Orville
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：December 4, 2007
• 年：2007
• 卷：46
• 期：48
• 页码：13800 - 13808
• 全文大小：416K
• 年卷期：v.46,no.48(December 4, 2007)
• ISSN：1520-4995

文摘

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondarynitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoAdehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removingthe substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts asthe base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterizedkinetically and structurally. The mutation results in a decrease in the rate constant for proton abstractionof 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-raycrystallography to a resolution of 2.65 Å, shows that the critical structural change is an increase in thedistance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of theresidue at position 409. The D402E mutation results in a smaller decrease in the rate constant for protonabstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 Å resolution, shows thatthere is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and theinteraction of this residue with Ser276 is perturbed. These results establish the critical importance of theinteraction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.