Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction

Abstract

The flavoenzyme nitroalkane oxidase is a member of the acyl-CoA dehydrogenase superfamily. Nitroalkane oxidase catalyzes the oxidation of neutral nitroalkanes to nitrite and the corresponding aldehydes or ketones. Crystal structures to 2.2 Å resolution or better are described of enzyme complexes with bound substrates and of a trapped substrate-flavin adduct. The D402N enzyme has no detectable activity with neutral nitroalkanes (Valley, M. P., and Fitzpatrick, P. F. (2003) J. Am. Chem. Soc. 23,[8738][8739]. The structure of the D402N enzyme crystallized in the presence of 1-nitrohexane or 1-nitrooctane shows the presence of the substrate in the binding site. The aliphatic chain of the substrate extends into a tunnel leading to the enzyme surface. The oxygens of the substrate nitro group interact both with amino acid residues and with the 2'-hydroxyl of the FAD. When nitroalkane oxidase oxidizes nitroalkanes in the presence of cyanide, an electrophilic flavin imine intermediate can be trapped (Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) J. Am. Chem. Soc. 127, 2062-2066. The structure of the enzyme trapped with cyanide during oxidation of 1-nitrohexane shows the presence of the modified flavin. A continuous hydrogen bond network connects the nitrogen of the CN-hexyl-FAD through the FAD 2'-hydroxyl to a chain of water molecules extending to the protein surface. Together, our complementary approaches provide strong evidence that the flavin cofactor is in the appropriate oxidation state and correlates well with the putative intermediate state observed within each of the crystal structures. Consequently, these results provide important structural descriptions of several steps along the nitroalkane oxidase reaction cycle.Nitroalkane oxidase (NAO 1 ) from the soil fungus Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the corresponding aldehydes or ketones with the release of nitrite and the † This research was supported in part by grants to PFF from the NIH (GM058698) and The Welch Foundation (A-1245) and to AMO from the Offices of Biological and Environmental Research US Department of Energy, the National Center for Research Resources (2 P41 RR012408) of the NIH and from the of the US Department of Energy. 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. *Corresponding authors. PFF: phone, fax, fitzpatrick@biochem.uthscsa The atomic coordinates and structure factors have been deposited with the Protein Data Bank with the corresponding file names: a) D402N NAO plus 1-nitrohexane, 3D9D; b) D402N NAO plus 1-nitrooctane, 3D9E; c) S276A NAO plus 1-nitrohexane, 3D9F; and d) wild-type NAO containing the N5-cyanohexyl FAD, 3D9G. 1 Abbreviations: NAO, nitroalkane oxidase; ACO, acyl-CoA oxidase; ACAD, acyl-CoA dehydrogenase; K ne , steady-state K m for nitroethane.

A Switch between One- and Two-electron Chemistry of the Human Flavoprotein Iodotyrosine Deiodinase Is Controlled by Substrate

“…In these examples, hydrogen bonding provided by the 2Ј-hydroxyl group polarizes the acyl carbonyl and stabilizes its developing charge. Nitroalkane oxidase is a member of the same acyl-CoA dehydrogenase superfamily and demonstrates an equivalent interaction between the 2Ј-hydroxyl of FAD and an oxygen of the nitro group (42). This same activation strategy may also be proposed for BluB, an enzyme closely related to IYD in structure but not function (7,15).…”

Differential quantum tunneling contributions in nitroalkane oxidase catalyzed and the uncatalyzed proton transfer reaction

“…The variability likely results from a combination of real enzyme-substrate binding effects and the modest resolution of the crystal structure. For comparison, the crystal structures with the substrates 1-nitrohexane or 1-nitrooctane demonstrate similar complexes, but with less binding orientation variability (33). Thus, nitroethane exhibits the most active site variability in the D402N complex, which correlates well with the lower k cat /K m value relative to the longer chain nitroalkane substrates (7,8).…”

“…We have determined crystal structures of NAO complexes with a series of nitroalkanes (33) including nitroethane, which is reported here (Fig. 1A).…”

A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase

“…This flavin intermediate has not been previously observed for flavoprotein oxidases but has been limited to reactions of flavoprotein monooxygenases (5)(6)(7). Besides P2O, an intermediate in flavoprotein oxidases has been detected only in the crystalline forms of choline oxidase (1.86 Å) (8) and nitroalkane oxidase (in the presence of cyanide) (9), and in the mutant form, C42S, of an NADH oxidase (10). In the case of glucose oxidase from Aspergillus niger, the generation of a flavin semiquinone-superoxide radical pair using pulse radiolysis resulted in formation of a putative C4a-hydroperoxyflavin intermediate (11).…”