Mariliz Ortiz-Maldonado

Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.

para-Hydroxybenzoate hydroxylase catalyzes a two-step reaction that demands precise control of solvent access to the catalytic site. The first step of the reaction, reduction of flavin by NADPH, requires access to solvent. The second step, oxygenation of reduced flavin to a flavin C4a-hydroperoxide that transfers the hydroxyl group to the substrate, requires that solvent be excluded to prevent breakdown of the hydroperoxide to oxidized flavin and hydrogen peroxide. These conflicting requirements are met by the coordination of multiple movements involving the protein, the two cofactors, and the substrate. Here, using the R220Q mutant form of para-hydroxybenzoate hydroxylase, we show that in the absence of substrate, the large βαβ domain (residues 1–180) and the smaller sheet domain (residues 180–270) separate slightly, and the flavin swings out to a more exposed position to open an aqueous channel from the solvent to the protein interior. Substrate entry occurs by first binding at a surface site and then sliding into the protein interior. In our study of this mutant, the structure of the complex with pyridine nucleotide was obtained. This cofactor binds in an extended conformation at the enzyme surface in a groove that crosses the binding site of FAD. We postulate that for stereospecific reduction, the flavin swings to an out position and NADPH assumes a folded conformation that brings its nicotinamide moiety into close contact with the isoalloxazine moiety of the flavin. This work clearly shows how complex dynamics can play a central role in catalysis by enzymes.

Studies on the Mechanism of p-Hydroxyphenylacetate 3-Hydroxylase from Pseudomonas aeruginosa – a System Composed of a Small Flavin Reductase and a Large Flavin-Dependent Oxygenase

There are two known types of microbial two-component flavin-dependent monooxygenases that catalyze oxygenation of p-hydroxyphenylacetate (HPA), and they are distinguished by having structurally distinct reductases and oxygenases. This paper presents a detailed analysis of the properties of the enzyme from Pseudomonas aeruginosa, an example of one group, and compares its properties to those published for the Acinetobacter baumannii enzyme, an example of the alternative group. The reductase and oxygenase from P. aeruginosa were expressed in Escherichia coli. The reductase was purified as a stable C-terminally His-tagged yellow protein containing weakly bound FAD, and the oxygenase was purified as a stable colorless N-terminally His-tagged protein. The reductase catalyzes the reduction of FAD by NADH and releases the FADH(-) product into solution, but unlike the reductase from A. baumannii, this catalysis is not influenced by HPA. The oxygenase binds the released FADH(-) and catalyzes the oxygenation of HPA to form 3,4-dihydroxyphenylacetate, after which the FAD dissociates to be re-reduced by the reductase, a common overall pattern for two-component flavin-dependent oxygenases. With this system, it appears that interactions between the reductase and the oxygenase can facillitate the transfer of FADH(-) to the oxygenase, although they are not required. We show that the P. aeruginosa oxygenase system in complex with FADH(-) reacts with O(2) to form a quasi-stable, unusually high-extinction flavin hydroperoxide species that binds HPA and reacts to form the product. The resultant flavin hydroxide decomposes to FAD and water while still bound to the oxygenase and then releases product and FAD from the protein. Unlike the enzyme from A. baumannii, during normal catalysis involving both the reductase and oxygenase, the rate-determining step in catalysis is the dissociation of FAD from the oxygenase in a process that is independent of the concentration of HPA. Structures for the reductases and oxygenases from A. baumannii and from Thermus thermophilus (similar to the P. aeruginosa system) form a basis for interpreting the molecular origins of the differences between the two groups of flavin-dependent two-component oxygenases.

Detection of intermediates in the oxidative half-reaction of the FAD-dependent thymidylate synthase from Thermotoga maritima: carbon transfer without covalent pyrimidine activation.

Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine 5′-monophosphate (dUMP) and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF), synthesizing 2′-deoxythymidine 5′-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH2THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis.