scholarly journals Characterization of coenzyme binding and selectivity determinants in Mycobacterium tuberculosis flavoprotein reductase A: analysis of Arg199 and Arg200 mutants at the NADP(H) 2′-phosphate binding site

2008 ◽  
Vol 417 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Muna Sabri ◽  
Adrian J. Dunford ◽  
Kirsty J. McLean ◽  
Rajasekhar Neeli ◽  
Nigel S. Scrutton ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Substrate Inhibition ◽  
Reduction Rate ◽  
Binding Mode ◽  
Phosphate Binding ◽  
Ferricyanide Reduction ◽  
Coenzyme Specificity ◽  
Coenzyme Binding ◽  
Kd Values ◽  
Adrenodoxin Reductase

Mycobacterium tuberculosis FprA (flavoprotein reductase A) is an NAD(P)H- and FAD-binding reductase that is structurally/evolutionarily related to adrenodoxin reductase. Structural analysis implicates Arg199 and Arg200 in interactions with the NADP(H) 2′-phosphate group. R199A, R200A and R199A/R200A mutants were characterized to explore the roles of these basic residues. All mutations abolished neutral FAD semiquinone stabilization in the NADPH-reduced enzyme, owing to weakened NADPH affinity. Instead, FAD hydroquinone was formed in all mutants, and each displayed substantially enhanced autooxidation rates (20–40-fold) compared with NADPH-reduced WT (wild-type) FprA. Steady-state ferricyanide reduction studies revealed diminished NADPH affinity (higher Km values), but lower NADH Km values. Despite a lowered kcat, the R199A/R200A mutant exhibited a 200-fold coenzyme specificity switch towards NADH, although substrate inhibition was observed at high NADH concentrations (Ki=250 μM). Stopped-flow FAD reduction studies confirmed substantially increased NADPH Kd values, although the limiting flavin reduction rate constant was similar in all mutants. The R199A mutation abolished electron transfer between hydroquinone FprA and NADP+, while this reaction progressed (via an FADH2-NADP+ charge-transfer intermediate) for R200A FprA, albeit more slowly (klim=58.1 s−1 compared with >300 s−1) than in WT. All mutations caused positive shifts in FAD potential (∼40–65 mV). Binding of an NADPH analogue (tetrahydro-NADP) induced negative shifts in potential (∼30–40 mV) only for variants with the R200A mutation, indicating distinctive effects of Arg199/Arg200 on coenzyme binding mode and FAD potential. Collectively, these data reveal important roles for the phylogenetically conserved arginines in controlling FprA FAD environment, thermodynamics, coenzyme selectivity and reactivity.

scholarly journals Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA

2003 ◽  
Vol 372 (2) ◽  
pp. 317-327 ◽  
Author(s):  
Kirsty J. McLEAN ◽  
Nigel S. SCRUTTON ◽  
Andrew W. MUNRO
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cytochrome P450 ◽  
Spectral Characteristics ◽  
Reduction Rate ◽  
Pyridine Nucleotide ◽  
Cytochrome P450 Enzymes ◽  
Hydrogen Electrode ◽  
Ferricyanide Reduction ◽  
Ferredoxin Reductase ◽  
Adrenodoxin Reductase

The genome sequence of the pathogenic bacterium Mycobacterium tuberculosis revealed numerous cytochrome P450 enzymes, which require accessory redox enzymes for catalytic function (ferredoxin reductase and ferredoxin). The most likely ferredoxin reductase is encoded by fprA, and its structure resembles eukaryotic adrenodoxin reductases. We have cloned, expressed and purified the flavoenzyme product of the fprA gene in Escherichia coli. FprA reduces various electron acceptors using either NADPH or NADH as the electron donor, but discriminates in favour of NADPH (apparent Km for NADH=50.6±3.1 μM; NADPH=4.1±0.3 μM from ferricyanide reduction experiments). Stopped-flow studies of reduction of the FprA FAD by NADPH demonstrate increased flavin reduction rate at low NADPH concentration (<200 μM), consistent with the presence of a second, kinetically distinct and inhibitory, pyridine nucleotide-binding site, similar to that identified in human cytochrome P450 reductase [Gutierrez, Lian, Wolf, Scrutton and Roberts (2001) Biochemistry 40, 1964–1975]. Flavin reduction by NADH is slower than with NADPH and displays hyperbolic dependence on NADH concentration [maximal reduction rate (kred)=25.4±0.7 s−1, apparent Kd=42.9±4.6 μM]. Flavin reoxidation by molecular oxygen is more rapid for NADH-reduced enzyme. Reductive titrations show that the enzyme forms a species with spectral characteristics typical of a neutral (blue) FAD semiquinone only on reduction with NADPH, consistent with EPR studies. The second order dependence of semiquinone formation on the concentration of FprA indicates a disproportionation reaction involving oxidized and two-electron-reduced FprA. Titration of FprA with dithionite converts oxidized FAD into the hydroquinone form; the flavin semiquinone is not populated under these conditions. The midpoint reduction potential for the two electron couple is −235±5 mV (versus the normal hydrogen electrode), similar to that for adrenodoxin reductase (−274 mV). Our data provide a thermodynamic and transient kinetic framework for catalysis by FprA, and complement recent spectrophotometric and steady-state studies of the enzyme [Fischer, Raimondi, Aliverti and Zanetti (2002) Eur. J. Biochem. 269, 3005–3013].

scholarly journals The –SH Protection Method for Determining Accurate Kd Values for Enzyme-Coenzyme Complexes of NAD+-Dependent Glutamate Dehydrogenase and Engineered Mutants: Evidence for Nonproductive NADPH Complexes

2010 ◽  
Vol 2010 ◽  
pp. 1-5 ◽  
Author(s):  
Joanna Griffin ◽  
Paul C. Engel
Keyword(s):  
Glutamate Dehydrogenase ◽  
Binding Mode ◽  
Apparent Contradiction ◽  
Double Mutation ◽  
Coenzyme Binding ◽  
Protection Method ◽  
Kd Values ◽  
Activity Measurements ◽  
Nonproductive Binding

Inactivation rates have been measured for clostridial glutamate dehydrogenase and several engineered mutants at various DTNB concentrations. Analysis of rate constants allowed determination of Kd for each non-covalent enzyme-DTNB complex and the rate constant for reaction to form the inactive enzyme-thionitrobenzoate adduct. Both parameters are sensitive to the mutations F238S, P262S, the double mutation F238S/P262S, and D263K, all in the coenzyme binding site. Study of the effects of NAD+, NADH and NADPH at various concentrations in protecting against inactivation by 200 μM DTNB allowed determination of Kd values for binding of these coenzymes to each protein, yielding surprising results. The mutations were originally devised to lessen discrimination against the disfavoured coenzyme NADP(H), and activity measurements showed this was achieved. However, the Kd determinations indicated that, although Kd values for NAD+ and NADH were increased considerably, Kd for NADPH was increased even more than for NADH, so that discrimination against binding of NADPH was not decreased. This apparent contradiction can only be explained if NADPH has a nonproductive binding mode that is not weakened by the mutations, and a catalytically productive mode that, though strengthened, is masked by the nonproductive binding. Awareness of the latter is important in planning further mutagenesis.

scholarly journals Design of potential bisubstrate inhibitors against Mycobacterium tuberculosis (Mtb) 1-deoxy-d-xylulose 5-phosphate reductoisomerase (Dxr)—evidence of a novel binding mode

MedChemComm ◽  
2013 ◽  
Vol 4 (7) ◽  
pp. 1099 ◽  
Author(s):  
Géraldine San Jose ◽  
Emily R. Jackson ◽  
Eugene Uh ◽  
Chinchu Johny ◽  
Amanda Haymond ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Mode ◽  
Bisubstrate Inhibitors

scholarly journals Effect of ionophores on carrier-mediated electron translocation in ferricyanide-containing liposomes

1979 ◽  
Vol 184 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Mette Miller ◽  
Lars Chr. Petersen ◽  
Finn B. Hansen ◽  
Peter Nicholls
Keyword(s):  
Reduction Rate ◽  
Electron Donors ◽  
Oxidation Products ◽  
Ph Gradient ◽  
Ferricyanide Reduction ◽  
Electron Carrier ◽  
Carbonyl Cyanide ◽  
Biochemical Systems ◽  
Stimulation Of

Ferricyanide-containing liposomes were used as a system to compare the electron- and proton-translocating properties of six redox reagents commonly used as electron donors for biochemical systems. The effects of different ionophore combinations on the ferricyanide-reduction rate were generally consistent with the expected proton- and electron-translocating properties of the mediators. The transmembrane pH gradient produced by hydrogen carriers was demonstrated. Nigericin or valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone are capable of collapsing this gradient and of stimulating ferricyanide reduction mediated by this type of carrier. No pH gradient is produced with the electron carrier 1,1′-dibutylferrocene. In the presence of tetraphenylboron anion, which is needed for this carrier to act as an efficient mediator, addition of valinomycin alone is sufficient to obtain full stimulation of ferricyanide reduction. NNN′N′-Tetramethyl-p-phenylenediamine does not behave as a simple electron carrier. During NNN′N′-tetramethyl-p-phenylenediamine-mediated ferricyanide reduction protons are translocated across the membrane and accumulated in the vesicles. This is not due to the presence of demethylated impurities in the NNN′N′-tetramethyl-p-phenylenediamine sample, but may be the result of an accumulation of oxidation products other than the Wurster's Blue radical. These results suggest a reconsideration of studies on protonmotive forces across membranes where NNN′N′-tetramethyl-p-phenylenediamine is used as a mediator.

Elucidation of the coenzyme binding mode of further B12-dependent enzymes using a base-off analogue of coenzyme B12

2000 ◽  
Vol 10 (1-3) ◽  
pp. 345-350 ◽  
Author(s):  
László Poppe ◽  
Harald Bothe ◽  
Gerd Bröker ◽  
Wolfgang Buckel ◽  
Erhard Stupperich ◽  
...  
Keyword(s):  
Binding Mode ◽  
Coenzyme B12 ◽  
Coenzyme Binding

scholarly journals Engineering the nucleotide coenzyme specificity and sulfhydryl redox sensitivity of two stress-responsive aldehyde dehydrogenase isoenzymes of Arabidopsis thaliana

2011 ◽  
Vol 434 (3) ◽  
pp. 459-471 ◽  
Author(s):  
Naim Stiti ◽  
Isaac O. Adewale ◽  
Jan Petersen ◽  
Dorothea Bartels ◽  
Hans-Hubert Kirch
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Structures ◽  
Aromatic Aldehydes ◽  
Carbon Chain ◽  
Cysteine Residues ◽  
Carbon Chain Length ◽  
Coenzyme Specificity ◽  
Coenzyme Binding ◽  
Catalytic Cysteine ◽  
Binding Cleft

Lipid peroxidation is one of the consequences of environmental stress in plants and leads to the accumulation of highly toxic, reactive aldehydes. One of the processes to detoxify these aldehydes is their oxidation into carboxylic acids catalyzed by NAD(P)+-dependent ALDHs (aldehyde dehydrogenases). We investigated kinetic parameters of two Arabidopsis thaliana family 3 ALDHs, the cytosolic ALDH3H1 and the chloroplastic isoform ALDH3I1. Both enzymes had similar substrate specificity and oxidized saturated aliphatic aldehydes. Catalytic efficiencies improved with the increase of carbon chain length. Both enzymes were also able to oxidize α,β-unsaturated aldehydes, but not aromatic aldehydes. Activity of ALDH3H1 was NAD+-dependent, whereas ALDH3I1 was able to use NAD+ and NADP+. An unusual isoleucine residue within the coenzyme-binding cleft was responsible for the NAD+-dependence of ALDH3H1. Engineering the coenzyme-binding environment of ALDH3I1 elucidated the influence of the surrounding amino acids. Enzyme activities of both ALDHs were redox-sensitive. Inhibition was correlated with oxidation of both catalytic and non-catalytic cysteine residues in addition to homodimer formation. Dimerization and inactivation could be reversed by reducing agents. Mutant analysis showed that cysteine residues mediating homodimerization are located in the N-terminal region. Modelling of the protein structures revealed that the redox-sensitive cysteine residues are located at the surfaces of the subunits.

scholarly journals Elucidating the Phosphate Binding Mode of Phosphate-Binding Protein: The Critical Effect of Buffer Solution

2018 ◽  
Vol 122 (24) ◽  
pp. 6371-6376 ◽  
Author(s):  
Rui Qi ◽  
Zhifeng Jing ◽  
Chengwen Liu ◽  
Jean-Philip Piquemal ◽  
Kevin N. Dalby ◽  
...  
Keyword(s):  
Binding Protein ◽  
Binding Mode ◽  
Phosphate Binding ◽  
Buffer Solution ◽  
Critical Effect ◽  
Phosphate Binding Protein
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