Joint functions of protein residues and NADP(H) in oxygen activation by flavin-containing monooxygenase

Abstract

The reactivity of flavoenzymes with dioxygen is at the heart of a number of biochemical reactions with far reaching implications for cell physiology and pathology. Flavin-containing monooxygenases are an attractive model system to study flavin-mediated oxygenation. In these enzymes, the NADP(H) cofactor is essential for stabilizing the flavin intermediate, which activates dioxygen and makes it ready to react with the substrate undergoing oxygenation. Our studies combine site-directed mutagenesis with the usage of NADP(+) analogues to dissect the specific roles of the cofactors and surrounding protein matrix. The highlight of this "double-engineering" approach is that subtle alterations in the hydrogen bonding and stereochemical environment can drastically alter the efficiency and outcome of the reaction with oxygen. This is illustrated by the seemingly marginal replacement of an Asn to Ser in the oxygen-reacting site, which inactivates the enzyme by effectively converting it into an oxidase. These data rationalize the effect of mutations that cause enzyme deficiency in patients affected by the fish odor syndrome. A crucial role of NADP(+) in the oxygenation reaction is to shield the reacting flavin N5 atom by H-bond interactions. A Tyr residue functions as backdoor that stabilizes this crucial binding conformation of the nicotinamide cofactor. A general concept emerging from this analysis is that the two alternative pathways of flavoprotein-oxygen reactivity (oxidation versus monooxygenation) are predicted to have very similar activation barriers. The necessity of fine tuning the hydrogen-bonding, electrostatics, and accessibility of the flavin will represent a challenge for the design and development of oxidases and monoxygenases for biotechnological applications.

FIGURE 1.

Properties of FMO enzymes. A, overall scheme of the reaction and chemical structure of the crucial flavin-hydroperoxide intermediate that results from the reaction of dioxygen with the C4a atom of the reduced flavin. Stabilization of this intermediate requires the presence of a bound NADP⁺ molecule. Therefore, NADP(H) must have two binding modes; one competent in flavin reduction by hydride transfer and one competent in intermediate stabilization (13). B, overall three-dimensional structure of mFMO bound to NADP⁺. The NADP-binding and FAD-binding domains are in blue and cyan, respectively. Nitrogens are in blue, oxygens in red, phosphorous atoms in brown, flavin carbons in yellow, and NADP⁺ carbons in cyan.

FIGURE 2.

Presteady-state investigation of mFMO. A, spectra of deconvoluted enzyme species observed during the oxidative half-reaction in the (A) wild-type and (B) N78S proteins. Anerobic enzymes (12–15 μm protein in 50 mm Tris/HCl, pH 8.0, 100 mm NaCl, 5% glycerol, 3 mm β-mercaptoethanol) solutions were anaerobically reduced with NADPH and subsequently mixed with oxygenated buffer (125 μm oxygen). Numerical integrations (Pro-K software, Applied Photophysics Ltd) yielded a three-step model for wild-type protein in which the first step reflects the formation of the flavin-hydroperoxide (A → B = 8 s⁻¹). The rates for the B → C and C → D are 4 s⁻¹ and 0.5 s⁻¹, respectively. Their values are independent of oxygen concentrations. The analogous kinetic data of N78S mutant were best fitted using a single-step model (Table 1).

FIGURE 3.

Structural data on mFMO mutants. The coloring is as in Fig. 1B with the carbons of highlighted residues in green. A, structure of N78S mutation does not perturb the protein conformation. The picture shows the superposition of the N78S structure onto that of the wild-type enzyme in the same orientation as in Fig. 1B. The superposition yields a root-mean-square deviation of 0.16 Å for 443 Cα atoms (with reference to monomer A of the tetrameric enzyme). For the sake of clarity, only the side chain of Ser-78 of the mutant enzyme is shown (carbons in green). The wild-type structure is colored as in Fig. 1B. The carboxamide group of NADP⁺ is oriented with its -NH₂ group pointing toward the flavin ring as to make H-bonds (dashed lines) with the flavin N5 and O4 atoms. We cannot fully rule out that upon flavin reduction, the carboxamide flips so that its carbonyl oxygen (rather than the amide) would point toward the flavin. However, this would cause an unfavorable short contact between the NADP⁺ oxygen and flavin O4 and between the NADP⁺ nitrogen and Arg-413. B, structure of the N78D mutant exhibits an altered NADP⁺ binding mode with the nicotinamide ring flipped out toward the surface. This alteration does not involve any large conformational change in the protein (root-mean-square deviation of 0.37 Å). The coloring is as in Fig. 1B with the carbons of Asp-78 in green. C, N78K mutant displays a disordered nicotinamide ring (which is therefore not shown) and Tyr-212 oriented toward the flavin to partly occupy the nicotinamide site. To illustrate the underlying conformational change, the Tyr-212 side chain and NADP⁺ in the positions as observed in the wild-type enzyme are shown as black thin sticks. The terminal oxygen of Lys-78 is not fully ordered in the crystal structure. It might form a H-bond with the NADP⁺ ribose.

FIGURE 4.

Binding of NADP⁺ analogues to mFMO. A, chemical formulas of the two compounds using for this study (APADP⁺ on the left and thioNADP⁺ on the right). B, structure of the complex with APADP⁺ in approximately the same orientation and coloring scheme as in Fig. 1B. The carbons of APADP⁺ are in green. H-bonds are shown as dashed lines. C, comparison between the binding of NADP⁺ and APADP⁺ using a composite picture that shows the protein surface of the wild-type protein bound to NADP⁺ (carbons in cyan) and FAD (carbons in yellow). The pyridine ring of APADP⁺ is superimposed to highlight its different position at the rim of the active-site cleft surface. The NADP⁺ and APADP⁺ complexes are very similar (root-mean-square deviation of 0.21 Å for the Cα atoms).