Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

The proton transfer reaction between the substrate nitroethane and Asp-402 catalyzed by nitroalkane oxidase and the uncatalyzed process in water have been investigated using a path-integral free-energy perturbation method. Although the dominating effect in rate acceleration by the enzyme is the lowering of the quasiclassical free energy barrier, nuclear quantum effects also contribute to catalysis in nitroalkane oxidase. In particular, the overall nuclear quantum effects have greater contributions to lowering the classical barrier in the enzyme, and there is a larger difference in quantum effects between proton and deuteron transfer for the enzymatic reaction than that in water. Both experiment and computation show that primary KIEs are enhanced in the enzyme, and the computed Swain-Schaad exponent for the enzymatic reaction is exacerbated relative to that in the absence of the enzyme. In addition, the computed tunneling transmission coefficient is approximately three times greater for the enzyme reaction than the uncatalyzed reaction, and the origin of the difference may be attributed to a narrowing effect in the effective potentials for tunneling in the enzyme than that in aqueous solution.PI-FEP/UM simulations ͉ enzyme catalysis ͉ kinetic isotope effects ͉ X-ray structure A lthough the dominant factor in enzyme catalysis is the lowering of the quasiclassical free energy barrier of the enzymatic reaction in comparison with the uncatalyzed process (1-3), quantum mechanical tunneling has been recognized to also play a role in enzymatic hydrogen transfer reactions (2, 3). An intriguing, yet unanswered, question is whether enzymes have evolved to enhance tunneling to accelerate the reaction rate because quantum effects on rate acceleration are much smaller than hydrogen bonding and electrostatic stabilization of the transition state (1, 4, 5). Nevertheless, a small factor of two in rate enhancement can have important physiological impacts. Although it appears straightforward to address this question by comparing the enzymatic and the uncatalyzed reaction in solution, the difficulty is to design a model system that mimics exactly the same enzymatic reaction and mechanism. The present study examines the structure of nitroalkane oxidase (NAO) complex with nitroethane and kinetic isotope effects at the primary and secondary sites for the enzymatic and the uncatalyzed reaction. The computational findings are consistent with experimental data, suggesting that there is a differential tunneling effect for the proton transfer reaction in NAO and in water. Analysis of tunneling paths reveals that the enzyme reduces both the free energy of activation and the width of the effective potential, resulting in enhanced proton tunneling in the active site.The flavoenzyme NAO catalyzes the conversion of nitroalkanes to nitrite and aldehydes or ketones (Fig. S1) (6). The ␣-proton abstraction of the small substrate nitroethane by Asp-402 is rate-limiting, which is accelerated by a factor of 10 9 over the uncatalyzed reaction between n...

Novel bioluminogenic substrates were designed for probing monoamine oxidase (MAO) activity based on a simple and effective beta-elimination strategy. By modifying the amino group and the central core of luciferin derivatives, we have developed a series of substrates useful for assays of MAO A or B, or both. One of these substrates, exhibiting low Km values and high signal-to-background ratios with both isozymes, was shown to accurately measure the Ki values of known MAO inhibitors. This substrate is a key component in the development of a highly sensitive homogeneous MAO assay for high-throughput screening (HTS) of compounds in drug discovery and for monitoring MAO activity in complex biological systems. This design strategy should be applicable to fluorogenic MAO substrates and could broaden the structural requirements of substrates for other enzyme assays.

The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitroalkanes to the respective aldehydes or ketones, releasing nitrite. The enzyme has recently been identified as being homologous to the acyl-CoA dehydrogenase family of enzymes [Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2702-2707. The glutamate which acts as an active site base in that family of enzymes aligns with Asp402 of nitroalkane oxidase. To evaluate the identification of Asp402 as an active site base, the effect of mutation of Asp402 to glutamate on the rate of cleavage of the nitroalkane C-H bond has been determined. Deuterium kinetic isotope effects on steady state kinetic parameters and direct measurement of the rate of flavin reduction establish that the mutation increases the ΔG ‡ for C-H bond cleavage by 1.6-1.9 kcal/mol. There is no effect on the rate of reaction of the reduced enzyme with oxygen. These results support the assignment of Asp402 as the active site base in nitroalkane oxidase.The flavoenzyme nitroalkane oxidase (NAO) 1 catalyzes the oxidation of nitroalkanes to the respective aldehydes or ketones with consumption of oxygen and release of nitrite and hydrogen peroxide (Scheme 1) (1). While several other flavoprotein oxidases are able to oxidize nitroalkanes, they require the preformed anion as a substrate in a clearly nonphysiological reaction (2). NAO is unique in that it uses the neutral forms of nitroalkanes as substrates (3); moreover, the enzyme is induced by growth of Fusarium oxysporum on nitroethane, consistent with nitroalkane oxidation being the physiological role of the enzyme (1). NAO can oxidize a broad range of primary and secondary nitroalkanes, although primary aliphatic nitroalkanes are the best substrates (4).As is the case with many flavoproteins, the reaction of NAO can be divided into reductive and oxidative half-reactions (5). In the reductive half-reaction, the nitroalkane substrate reacts with the oxidized flavin to generate the oxidized product and reduced flavin. The mechanism of this half-reaction (Scheme 2) has been proposed to involve removal of the substrate α-hydrogen as a proton, followed by attack of the resulting carbanion on the flavin (6). Loss of nitrite from the resulting adduct would generate an electrophilic species to which hydroxide adds (path a); this flavin adduct then decomposes to form reduced flavin and aldehyde or ketone. Evidence for this mechanism comes from the demonstration that an inactive 5-nitrobutyl-FAD adduct

Nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones with the production of H(2)O(2) and nitrite. The flavoenzyme is a new member of the acyl-CoA dehydrogenase (ACAD) family, but it does not react with acyl-CoA substrates. We present the 2.2 A resolution crystal structure of NAO trapped during the turnover of nitroethane as a covalent N5-FAD adduct (ES*). The homotetrameric structure of ES* was solved by MAD phasing with 52 Se-Met sites in an orthorhombic space group. The electron density for the N5-(2-nitrobutyl)-1,5-dihydro-FAD covalent intermediate is clearly resolved. The structure of ES was used to solve the crystal structure of oxidized NAO at 2.07 A resolution. The c axis for the trigonal space group of oxidized NAO is 485 A, and there are six subunits (1(1)/(2) holoenzymes) in the asymmetric unit. Four of the active sites contain spermine (EI), a weak competitive inhibitor, and two do not contain spermine (E(ox)). The active-site structures of E(ox), EI, and ES* reveal a hydrophobic channel that extends from the exterior of the protein and terminates at Asp402 and the N5 position on the re face of the FAD. Thus, Asp402 is in the correct position to serve as the active-site base, where it is proposed to abstract the alpha proton from neutral nitroalkane substrates. The structures for NAO and various members of the ACAD family overlay with root-mean-square deviations between 1.7 and 3.1 A. The homologous region typically spans more than 325 residues and includes Glu376, which is the active-site base in the prototypical member of the ACAD family. However, NAO and the ACADs exhibit differences in hydrogen-bonding patterns between the respective active-site base, substrate molecules, and FAD. These likely differentiate NAO from the homologues and, consequently, are proposed to result in the unique reaction mechanism of NAO.