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The reactions of enzymes and cofactors with gaseous molecules such as dioxygen (O2) are challenging to study and remain among the most contentious subjects in biochemistry. To date, it is largely enigmatic how enzymes control and fine-tune their reactions with O2, as exemplified by the ubiquitous flavin-dependent enzymes that commonly facilitate redox chemistry such as the oxygenation of organic substrates. Here we employ O2-pressurized X-ray crystallography and quantum mechanical calculations to reveal how the precise positioning of O2 within a flavoenzyme's active site enables the regiospecific formation of a covalent flavin-oxygen adduct and oxygenating species (i.e., the flavin-N5-oxide) by mimicking a critical transition state. This study unambiguously demonstrates how enzymes may control the O2 functionalization of an organic cofactor as prerequisite for oxidative catalysis. Our work thus illustrates how O2 reactivity can be harnessed in an enzymatic environment and provides crucial knowledge for future rational design of O2-reactive enzymes.

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase to catalyse the methylation of the uracil moiety of 2'-deoxyuridine-5'-monophosphate. All known thymidylate synthases rely on an active site residue of the enzyme to activate 2'-deoxyuridine-5'-monophosphate (1,2). This functionality has been demonstrated for classical thymidylate synthases, including human thymidylate synthase, and is instrumental in mechanism-based inhibition of these enzymes. Here we report an example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens (3-5). Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (that is, a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2'-deoxythymidine-5'-monophosphate. These observations indicate a very different chemical cascade than that of classical thymidylate synthases or any other known biologicalmethylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Because several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs., Classical thymidylate synthases, encoded by the thyA and TYMS genes, are present in most eukaryotes, including humans, and are frequently targeted by chemotherapeutic and antibiotic drugs. A recently discovered class [...]

Tetrahydrofolate is a ubiquitous [C.sub.1] carrier in many biosynthetic pathways in bacteria, importantly, in the biosynthesis of formylmethionyl [tRNA.sup.fMET], which is essential for the initiation of translation. The final step in the biosynthesis of tetrahydrofolate is carried out by the enzyme dihydrofolate reductase (DHFR). A search of the complete genome sequence of Helicobacter pylori failed to reveal any sequence that encodes DHFR. Previous studies demonstrated that the H. pylori dihydropteroate synthase gene folP can complement an Escherichia coli strain in which folA and folM, encoding two distinct DHFRs, are deleted. It was also shown that H. pylori FolP possesses an additional N-terminal domain that binds flavin mononucleotide (FMN). Homologous domains are found in FolP proteins of other microorganisms that do not possess DHFR. In this study, we demonstrated that H. pylori FolP is also a dihydropteroate reductase that derives its reducing power from soluble flavins, reduced FMN and reduced flavin adenine dinucleotide. We also determined the stoichiometry of the enzymebound flavin and showed that half of the bound flavin is exchangeable with the soluble flavins. Finally, site-directed mutagenesis of the most conserved amino acid residues in the N-terminal domain indicated the importance of these residues for the activity of the enzyme as a dihydropteroate reductase.

Titrations, kinetics, and crystallography method were used to study the mechanism of inhibitor binding to the Class 1A dihydroorotate dehydrogenases (DHOD) from Lactococcus lactis. The observation of crystal structures for both inhibitor complexes showed that 3,5-diOHB binds in the same orientation as orotate while 3,4-diOHB binds in a twisted orientation, enabling one of its phenolic oxygens to form a very strong hydrogen bond to an asparagine.

The class 1A enzyme dihyroorotate dehydrogenase from Lactococcus lactis is examined to understand mechanism of flavin reduction. It is found that oxidation of dihyroorotate (DHO) to orotate (OA) by this enzyme is concerted, where the active site cysteine acts as a classical general base.

The development of menadione chemistry had led to choice of 6-[2'-(3'-methyl)-1',4'-naphthoquinolyl]hexanoic acid, called [M.sub.5] as a potent reversible and uncompetitive inhibitor of both human and Plasmodium falciparum glutathione reductases. The synthesis and kinetic characterization of a fluoromethyl-[M.sub.5] analogue that acts as a mechanism-based inhibitor of both enzymes is described.

Dihydroorotate dehydrogenase (DHOD) from Escherichia coli is a monomeric membrane-associated flavoprotein that catalyzes the oxidation of dihydroorotate to orotate. By using confocal fluorescence spectroscopy on the highly fluorescent Tyr318Leu mutant, we studied the catalytic turnover of single enzyme molecules through the characteristic on-off fluorescence signal.

The multi-phasic time course reactions of Mycobacterium tuberculosis lipoamide dehydrogenase reveals the formation of a transient reduced flavin intermediate. The compound is highly unstable.

Research has been conducted on Pseudomonas aeruginosa's proline 293 of p-hydroxybenzoate hydroxylase. The peptide segment flexibility in catalysis has been investigated via the substitution of this hydroxylase with serine by site-directed mutagenesis

The mechanism of action of amadoriase I from Aspergillus sp. is investigated using stopped-flow kinetic studies, which involves a charge-transfer intermediate. Results reveal that the active configuration of the substrate is the beta-pyranose form.

The flavin reduction in dihydroorotate dehydrogenase of Escherichia coli by dihydroorotate results in a reduced flavin-orotate charge-transfer complex. The rate of reduction is pH dependent suggesting Ser175 to be the active-site base. Data indicate that quinone menadione is a good oxidizing substrate for the enzyme.

Results reveal that the reduction of His72Asn p-hydroxybenzoate hydroxylase is controlled by the protonation state of the substrate p-hydroxybenzoate, which in turn mediates flavin movement during conformational changes. Data indicate that the ionization/protonation of the substrate proceeds via hydronium or hydroxide reacting with the enzyme-ligand complex.

Complexes involving FAD bound to p-hydroxybenzoate hydroxylase have been studied by X-ray crystallography. Results indicate the ability of isoalloxazine ring system of adopting in two positions on the protein and the possibility to obtain Raman spectra for the flavin ring.

The role of the serine hydrogen bond in the function of the native p-hydroxybenzoate hydroxylase (PHBH) has been studied by mutating residue 212 to alanine. PHBH is an aromatic flavoprotein hydroxylase that catalyzes the hydroxylation of p-hydroxybenzoate to form 3,4-dihydroxybenzoate. Results indicate that the residue is involved in substrate binding. The dissociation constant for p-hydroxybenzoate is one order of magnitude higher than for the wild-type but the catalytic process remains the same.

The enzyme p-hydroxybenzoate hydroxylase (PHBH) has to bind an ionizable phenolic substrate so that the NADPH may effectively reduce the enzyme's flavin. In the presence of NADPH, ionization of the phenol constitutes a 'password' that allows movement of the flavin ring system toward the surface of the enzyme, where reduction takes place. The linkage of substrate deprotonation to flavin movement is a novel mode of molecular recognition in which the enzyme tests the suitability of aromatic substrates before committing to the catalytic cycle.

The functions of flavin motions in catalysis, specifically in the reduction reaction, were determined by utilizing 6-azido-FAD as a special probe and a photolabel. Findings revealed that the artificial flavin changes the normal functioning of the enzyme only by a minimal amount. Hence, conclusions obtained for the FAD WT are directly applicable to the natural enzymes. FAD movements were found to play a role in the binding of the aromatic substrate and the reduction of the flavin by the NADPH.

Raman difference spectroscopic study of para-hydroxybenzoate hydroxylase was performed to investigate the chemistries of the substrate and flavin ring system. The Raman data provided information on changes in the ligand upon binding and changes in the flavin ring system of the enzyme in the enzyme-substrate complex. Deep red excitation was used to avoid interference from fluorescence background.

A study mutated lysine 297 of p-hydroxybenzoate hydroxylase to the neutral methionine residue and characterized the properties of the mutant enzyme. Findings indicate that regions of positive potential in the active site facilitate an important substrate activation that is required for rapid hydroxylation. They are consistent with existing theories of flavin mobility and substrate activation and underscore the significance of charge in the function of p-hydroxybenzoate hydroxylase.

Mutations at position 220 change the stability of the conformational states of the flavin of p-hydroxybenzoate hydroxylase and thus lead to changes in enzyme catalysis. The R220K enzyme stabilizes the conformation with flavin 'out' more than the wild type (WT) enzyme does, which lowers the R220K enzyme's catalytic efficiency. Catalytic mechanisms of the mutant R220K enzyme and the WT enzymes differ, as with partial hydroxylation of pOHB by R220K.

The Thr410 amino acid of the Yersinia protein tyrosine phosphatase controls the enzymatic activity by stabilizing the transition state in the dephosphorylation step. Pre-steady-state kinetic studies show that replacement of the residue with a hydroxy group destabilizes the ground-state interactions. Deletion of the residue increases the pK(sub a) of the active site thiol. Thr410 is a conserved hyroxyl group and is the nearest proton donor to the enzyme's active site.

Site-directed mutagenic conversion of Pseudomonas aeruginosa Asn 300 to Asp induces several dynamic, catalytic and structural changes in p-hydroxybenzoate hydroxylase. Many electrostatic and hydrogen bonding reactions reduce the substrate pKa. A single active-site residue is involved in most or all catalytic cycle processes. The Asn300Asp mutant does not reduce substrate pKa and enhances the flavin C(4a)-hydroxide stability.

X-ray crystallography studies of Pseudomonas aeruginosa mutant p-hydroxybenzoate hydroxylases reveal that mutant forms are isomorphous with wild-type Pseudomonas fluorescens enzyme crystals. The flavin and residue positions are not modified in the Tyr385Phe and Tyr201Phe structures. The substitution of Asp for Asn300 causes the side chain to move away from flavin, and disrupts carboxamide group-flavin O2 atom interactions.

Size exclusion chromatography and small-angle X-ray scattering (SAXS) has shown that the flavin-binding LOV sensor domain has not undergone a large conformational change in response to photon absorption. The chemical reduction of the FMN cofactor of LovK has attenuated the light-dependent ATPase activity of the protein in vitro, which has shown that LovK has functioned as conditional photosensor that is regulated by the oxidative state of the cellular environment.