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Pseudomonas aeruginosa PAO1 D-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital L-serine biosynthesis and is a potential therapeutic target against P. aeruginosa. PaD2HGDH, which oxidizes D-malate as an alternative substrate, has been demonstrated to be a metallo flavoprotein that requires Zn2+ for activity. However, the role of Zn2+ in the enzyme has not been elucidated, making it difficult to rationalize why nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn2+ or Co2+ to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate limiting for the enzyme's turnover at pH 7.4 with either D2HG or D-malate with similar rate constants for both substrates, irrespective of whether Zn2+ or Co2+ was bound to the enzyme. The steady-state pL profiles of the kcat and kcat/Km values with D-malate demonstrate that Zn2+ mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn2+ as a cofactor required for substrate orientation and activation during enzymatic turnover. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa couples the oxidation of D-2-hydroxyglutarate (D2HG) to L-serine biosynthesis for survival, using D-2-hydroxyglutarate dehydrogenase from P. aeruginosa (PaD2HGDH). Knockout of PaD2HGDH impedes P. aeruginosa growth, making PaD2HGDH a potential target for therapeutics. Previous studies showed that the enzyme's activity increased with Zn2+, Co2+, or Mn2+ but did not establish the enzyme's metal composition and whether the metal is an activator or a required cofactor for the enzyme, which we addressed in this study. Comparable to the human enzyme, PaD2HGDH showed only 15% flavin reduction with D2HG or D-malate. Upon purifying PaD2HGDH with 1 mM Zn2+, the Zn2+:protein stoichiometry was 2:1, yielding an enzyme with -40 s-1 kcat for D-malate. Treatment with 1 mM EDTA decreased the Zn2+:protein ratio to 1:1 without changing the kinetic parameters with D-malate. We observed complete enzyme inactivation for the metalloapoenzyme with 100 mM EDTA treatment, suggesting that Zn2+ is essential for PaD2HGDH activity. The presence of Zn2+ increased the flavin N3 atom pKa value to 11.9, decreased the flavin e450 at pH 7.4 from 13.5 to 11.8 mM-1 cm-1, and yielded a charged transfer complex with a broad absorbance band >550 nm, consistent with a Zn2+-hydrate species altering the electronic properties of the enzyme-bound FAD. The exogenous addition of Zn2+, Co2+, Cd2+, Mn2+, or Ni2+ to the metalloapoenzyme reactivated the enzyme in a sigmoidal pattern, consistent with an induced fit rapid-rearrangement mechanism. Collectively, our data demonstrate that PaD2HGDH is a Zn2+-dependent metallo flavoprotein, which requires Zn2+ as an essential cofactor for enzyme activity. [ABSTRACT FROM AUTHOR]

This review article describes the occurrence, general properties, and substrate specificity of the flavoenzymes belonging to the glucose-methanol-choline oxidoreductase superfamily and the l-α-hydroxyacid dehydrogenase family. Most of these enzymes catalyze the oxidations of hydroxyl groups, yielding carbonyl moieties. Over the years, carbanion, hydride transfer, and radical mechanisms have been discussed for these enzymes, and the main experimental evidences supporting these mechanisms are presented here. Regardless of the chemical nature of the organic substrate (i.e., activated and non-activated alcohols), a hydride transfer mechanism appears to be the most plausible for the flavoenzymes acting on CH-OH groups. The reaction of most of these enzymes likely starts with proton abstraction from the substrate hydroxyl group by a conserved active site histidine. Among the different approaches carried out to determine the chemical mechanisms with physiological substrates, primary substrate and solvent deuterium kinetic isotope effect studies have provided the most unambiguous evidences. It is expected that the numerous studies reported for these enzymes over the years will be instrumental in devising efficient industrial biocatalysts and drugs.

A new strategy is described for creating [Ca.sup.2} sensors by engineering a [Ca.sup.2} binding motif into sensitive locations of a fluorescent protein without the use of fluorescence resonance energy transfer (FRET) pairs. The developed [Ca.sup.2} sensor successfully targeted to the endoplasmic reticulum of mammalian cell lines to monitor [Ca.sup.2} changes occurring in response to stimulation with agonists.

The effects of pH and temperature on the substrate kinetic isotope effects with 1,2[sup.2.H.sub.4] choline is determined. The data indicates that the kinetic complexity arising from the net flux through the reverse of the hydride transfer step changed with temperature.

The mechanistic properties of the enzyme 2-nitropropane dioxygenase re investigated with solvent viscosity, pH and kinetic isotope effects. The data suggest that enzymatic turnover of 2-nitropropane dioxygenase with neutral substrates is limited by the cleavage of the substrate CH bond at low pH, whereas that with anionic substrates is limited by the non-oxidative tautomerization of ethylnitronate to nitroethane at high pH.

Research has been conducted on the nitroalkane oxidase that catalyzes nitroalkane oxidation to aldehydes or ketones. The investigation of the pH and kinetic isotope effects is described.

Research has been conducted on the flavoprotein nitroalkane oxidase. The kinetic mechanism of the active flavin adenine -containing enzyme form has been investigated.

Results demonstrate that nitroalkane oxidase contains a tyrosine residue in its active site and it may have a role in the substrate binding. Data discuss kinetic and structural characterization of the enzyme inactivated by tyrosine-specific tetranitromethane.

2-Oxo-3-pentynoate was found to be an active-site-directed inhibitor of certain flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed manner. The addition of FAD provides no protection from inactivation, while the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The rate of inactivation indicates saturation kinetics, consistent with the production of a reversible complex between the alkylating agent and the enzyme before inactivation occurs.

A study was conducted to characterize the active flavin adenine dinucleotide (FAD)-containing structure of nitroalkane oxidase from Fusarium oxysporum. Dialysis was carried out to prepare the apoprotein of nitroalkane oxidase while the molecular mass of enzymes was determined by MALDI-TOF mass spectrometry. Results indicated no catalytic activity when FAD was replaced with 5-deaza-FAD. Findings also showed that the features of FAD in nitroalkane oxidase were within the range associated for other oxidases.