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

Abstract

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homolog of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 Å, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively-charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 Å resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.The flavoenzyme nitroalkane oxidase (NAO) catalyzes the oxidation of nitroalkanes to the corresponding aldehydes or ketones with consumption of molecular oxygen and release of nitrite and hydrogen peroxide (Scheme 1) (1). NAO is unusual in that it catalyzes substrate oxidation by removing a substrate proton to form a carbanion intermediate (1,2), in contrast to † This research was supported in part by grants to PFF from the NIH (GM58698) and The Welch Foundation (A-1245) and to AMO from the American Heart Association (Grant in Aid 0555286B), the Offices of Biological and Environmental Research of the US Department of Energy, and the NIH (2 P41 RR012408). Use of the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the U.S. Department of Energy Office of Basic Energy Sciences, under Contract DE-AC02-98CH10886. ‡ The atomic coordinates and structure factors for NAO D402E and R409K have been deposited with the Protein Data Bank with the corresponding file names 2REH, 2rehsf, 2ZAF and rcsb027723, respectively. *Corresponding authors. PFF: phone, fax,; e-mail, fitzpat@tamu.edu. AMO: phone 631-344-4739; e-mail: amorv@bnl.gov. 1 Abbreviations: NAO, nitroalkane oxidase; ACAD, acyl-CoA dehydrogenase; K ne , the K m value for nitroethane as the substrate 2 For comparison, we examined Lys359, which is near Lys409 but is part of helix I. The Lys359 N□ atom forms ionic interactions with the backbone carbonyl oxygen atoms of residues Tyr398, Phe401, Gly403 and the OD1 atom of Asn405. The elect...

Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

“…In the active site of NAO, Asp402 interacts with two other residues, Arg409 and Ser276. The importance of Arg409 for proton abstraction from the substrate by Asp402 has been confirmed by site-directed mutagenesis (13). The rate constant for CH bond cleavage by the R409K enzyme is only 1% of the wild-type value.…”

“…The D k cat value for the wild-type enzyme is close to one because product release is substantially slower than the reductive half-reaction and consequently limits the overall turnover (9); this isotope effect increases in the S276A enzyme. Since the k cat value for NAO is simply a combination of the rate constants for flavin reduction and product release (3), the k cat and D k cat values can be used to calculate the rate constants for these two steps (13). The data in Table 2 yield a value of 6.3 ±1.2 s −1 for the rate constant for flavin reduction and a value of 1.2 ± 0.4 s −1 for the rate constant for product release.…”

Characterization of active site residues of nitroalkane oxidase

“…The active site base of NAO, Asp402, is part of a catalytic triad that also contains Arg409 and Ser276 (Figure 1). Mutation of any of these three residues decreases the rate constant for removal of the substrate proton by 2–3 orders of magnitude, confirming their importance 9,16,17. In addition, the structures of both the resting form of the enzyme and of the cyanide-trapped intermediate show that the hydroxyl of Serl71 forms a hydrogen bond with the N5 position of the flavin, suggesting it may modulate the reactivity of the flavin.…”

Identification of a Hypothetical Protein from Podospora anserina as a Nitroalkane Oxidase

“…The distance between Asp402 and Arg409 is appropriate for an electrostatic interaction, suggesting that Arg409 is involved in correctly positioning the active site base for catalysis. Mutation of Arg409 to lysine decreases the rate constant for proton abstraction by 100-fold, and alters the position of the active site base (20). The side chain hydroxyl of Ser276 forms hydrogen bonds with the carboxylate oxygens of Asp402 (20).…”

“…Mutation of Arg409 to lysine decreases the rate constant for proton abstraction by 100-fold, and alters the position of the active site base (20). The side chain hydroxyl of Ser276 forms hydrogen bonds with the carboxylate oxygens of Asp402 (20). Mutation of Ser276 to alanine decreases the rate of the proton abstraction by 100-fold without perturbing the active site structure (11).…”