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

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].

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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

Biochemical Journal ◽

10.1042/bj20080466 ◽

2008 ◽

Vol 417 (1) ◽

pp. 103-114 ◽

Cited By ~ 7

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.

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Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-09697-z ◽

2019 ◽

Vol 103 (9) ◽

pp. 3597-3614 ◽

Cited By ~ 9

Author(s):

Sandra Ortega Ugalde ◽

Maikel Boot ◽

Jan N. M. Commandeur ◽

Paul Jennings ◽

Wilbert Bitter ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cytochrome P450 ◽

Drug Targets ◽

Cytochrome P450 Enzymes ◽

Redox Partners

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Cytochrome P450 Alkane Hydroxylases of the CYP153 Family Are Common in Alkane-Degrading Eubacteria Lacking Integral Membrane Alkane Hydroxylases

Applied and Environmental Microbiology ◽

10.1128/aem.72.1.59-65.2006 ◽

2006 ◽

Vol 72 (1) ◽

pp. 59-65 ◽

Cited By ~ 202

Author(s):

Jan B. van Beilen ◽

Enrico G. Funhoff ◽

Alexander van Loon ◽

Andrea Just ◽

Leo Kaysser ◽

...

Keyword(s):

Cytochrome P450 ◽

Pseudomonas Putida ◽

Chain Length ◽

Nonheme Iron ◽

Cytochrome P450 Enzymes ◽

Medium Chain ◽

Ferredoxin Reductase ◽

Medium Chain Length ◽

Reductase Gene ◽

Alkane Hydroxylases

ABSTRACT Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.

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Loss of multiple enzyme activities due to the human genetic variation P284T in NADPH cytochrome P450 oxidoreductase

10.1101/643825 ◽

2019 ◽

Author(s):

Shaheena Parween ◽

Maria Natalia Rojas Velazquez ◽

Sameer S. Udhane ◽

Norio Kagawa ◽

Amit V. Pandey

Keyword(s):

Cytochrome P450 ◽

Cytochrome C ◽

Negative Impact ◽

Aromatase Activity ◽

Cytochrome P450 Enzymes ◽

Ferricyanide Reduction ◽

P450 Oxidoreductase ◽

Nadph Cytochrome ◽

Reduction Activity ◽

Cytochrome P450 Oxidoreductase

AbstractCytochromes P450 located in the endoplasmic reticulum require NADPH cytochrome P450 oxidoreductase (POR) for their catalytic activities. Mutations in POR cause multiple disorders in humans related to the biosynthesis of steroid hormones and also affect drug-metabolizing cytochrome P450 activities. Here we are reporting the effects of a POR genetic variant P284T which is located in the hinge region of POR that is necessary for the flexibility of domain movements. Human wild-type and P284T mutant of POR, as well as cytochrome P450 proteins, were expressed in bacteria, purified and then reconstituted in liposomes for enzyme kinetic assays. Quality of POR proteins was checked by cytochrome c, ferricyanide and tetrazolium dye reduction assay and measurements flavin content. We found that for the P284T variant of POR the cytochrome c reduction activity was reduced to 47% of the WT and MTT reduction was reduced to only 15% of the WT. No impact on ferricyanide reduction activity was observed, but a severe loss of CYP19A1 (aromatase) activity was observed (9% of WT). In the assays of drug metabolizing cytochrome P450 enzymes, the P284T variant of POR showed 26% activity for CYP2C9, 44% activity for CYP2C19, 23% activity for CYP3A4 and 44% activity in CYP3A5 assays compared to the WT POR. These results indicate a severe effect on several cytochrome P450 activities due to the P284T variation in POR which suggests a negative impact on both the steroid as well as drug metabolism in the individuals carrying this variation.

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Genetic and Mass Spectrometric Tools for Elucidating the Physiological Function(s) of Cytochrome P450 Enzymes from Mycobacterium tuberculosis

Methods in Molecular Biology - Cytochrome P450 Protocols ◽

10.1007/978-1-62703-321-3_7 ◽

2013 ◽

pp. 79-94 ◽

Cited By ~ 2

Author(s):

Hugues Ouellet ◽

Eric D. Chow ◽

Shenheng Guan ◽

Jeffery S. Cox ◽

Alma L. Burlingame ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Cytochrome P450 ◽

Physiological Function ◽

Mass Spectrometric ◽

Cytochrome P450 Enzymes

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Evaluation of Luminogenic Substrates as Probe Substrates for Bacterial Cytochrome P450 Enzymes: Application to Mycobacterium tuberculosis

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219853220 ◽

2019 ◽

Vol 24 (7) ◽

pp. 745-754

Author(s):

Sandra Ortega Ugalde ◽

Dongping Ma ◽

James J. Cali ◽

Jan N. M. Commandeur

Keyword(s):

Mycobacterium Tuberculosis ◽

Cytochrome P450 ◽

High Throughput ◽

High Throughput Screening ◽

Drug Targets ◽

High Signal ◽

Cytochrome P450 Enzymes ◽

High Enzyme ◽

Endogenous Substrates ◽

Selection Of

Several cytochrome P450 enzymes (CYPs) encoded in the genome of Mycobacterium tuberculosis (Mtb) are considered potential new drug targets due to the essential roles they play in bacterial viability and in the establishment of chronic intracellular infection. Identification of inhibitors of Mtb CYPs at present is conducted by ultraviolet-visible (UV-vis) optical titration experiments or by metabolism studies using endogenous substrates, such as cholesterol and lanosterol. The first technique requires high enzyme concentrations and volumes, while analysis of steroid hydroxylation is dependent on low-throughput analytical methods. Luciferin-based luminogenic substrates have proven to be very sensitive substrates for the high-throughput profiling of inhibitors of human CYPs. In the present study, 17 pro-luciferins were evaluated as substrates for Mtb CYP121A1, CYP124A1, CYP125A1, CYP130A1, and CYP142A1. Luciferin-BE was identified as an excellent probe substrate for CYP130A1, resulting in a high luminescence yield after addition of luciferase and adenosine triphosphate (ATP). Its applicability for high-throughput screening was supported by a high Z’-factor and high signal-to-background ratio. Using this substrate, the inhibitory properties of a selection of known inhibitors could be characterized using significantly less protein concentration when compared to UV-vis optical titration experiments. Although several luminogenic substrates were also identified for CYP121A1, CYP124A1, CYP125A1, and CYP142A1, their relatively low yield of luminescence and low signal-to-background ratios make them less suitable for high-throughput screening since high enzyme concentrations will be needed. Further structural optimization of luminogenic substrates will be necessary to obtain more sensitive probe substrates for these Mtb CYPs.

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Trp359 regulates flavin thermodynamics and coenzyme selectivity in Mycobacterium tuberculosis FprA

Biochemical Journal ◽

10.1042/bj20071298 ◽

2008 ◽

Vol 411 (3) ◽

pp. 563-570 ◽

Cited By ~ 4

Author(s):

Rajasekhar Neeli ◽

Muna Sabri ◽

Kirsty J. McLean ◽

Adrian J. Dunford ◽

Nigel S. Scrutton ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Catalytic Efficiency ◽

Tryptophan Residue ◽

Wild Type ◽

Hydrogen Electrode ◽

Visible Absorption ◽

Reduction Potentials ◽

Catalytic Function ◽

Adrenodoxin Reductase ◽

Fad Binding

Mtb (Mycobacterium tuberculosis) FprA (flavoprotein reductase A) is an NAD(P)H-dependent FAD-binding reductase that is structurally related to mammalian adrenodoxin reductase, and which supports the catalytic function of Mtb cytochrome P450s. Trp359, proximal to the FAD, was investigated in light of its potential role in controlling coenzyme interactions, as observed for similarly located aromatic residues in diflavin reductases. Phylogenetic analysis indicated that a tryptophan residue corresponding to Trp359 is conserved across FprA-type enzymes and in adrenodoxin reductases. W359A/H mutants of Mtb FprA were generated, expressed and the proteins characterized to define the role of Trp359. W359A/H mutants exhibited perturbed UV-visible absorption/fluorescence properties. The FAD semiquinone formed in wild-type NADPH-reduced FprA was destabilized in the W359A/H mutants, which also had more positive FAD midpoint reduction potentials (−168/−181 mV respectively, versus the standard hydrogen electrode, compared with −230 mV for wild-type FprA). The W359A/H mutants had lower ferricyanide reductase kcat and NAD(P)H Km values, but this led to improvements in catalytic efficiency (kcat/Km) with NADH as reducing coenzyme (9.6/18.8 μM−1·min−1 respectively, compared with 5.7 μM−1·min−1 for wild-type FprA). Stopped-flow spectroscopy revealed NAD(P)H-dependent FAD reduction as rate-limiting in steady-state catalysis, and to be retarded in mutants (e.g. limiting rate constants for NADH-dependent FAD reduction were 25.4 s−1 for wild-type FprA and 4.8 s−1/13.4 s−1 for W359A/H mutants). Diminished mutant FAD content (particularly in W359H FprA) highlighted the importance of Trp359 for flavin stability. The results demonstrate that the conserved Trp359 is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.

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Mycobacterium tuberculosis cytochrome P450 enzymes: a cohort of novel TB drug targets

Biochemical Society Transactions ◽

10.1042/bst20120062 ◽

2012 ◽

Vol 40 (3) ◽

pp. 573-579 ◽

Cited By ~ 19

Author(s):

Sean A. Hudson ◽

Kirsty J. McLean ◽

Andrew W. Munro ◽

Chris Abell

Keyword(s):

Mycobacterium Tuberculosis ◽

Cytochrome P450 ◽

High Throughput Screening ◽

Drug Targets ◽

Drug Resistant ◽

Cytochrome P450 Enzymes ◽

Front Line ◽

Exciting Potential ◽

Tuberculosis Disease ◽

Fragment Based Drug Discovery

TB (tuberculosis) disease remains responsible for the death of over 1.5 million people each year. The alarming emergence of drug-resistant TB has sparked a critical need for new front-line TB drugs with a novel mode of action. In the present paper, we review recent genomic and biochemical evidence implicating Mycobacterium tuberculosis CYP (cytochrome P450) enzymes as exciting potential targets for new classes of anti-tuberculars. We also discuss HTS (high-throughput screening) and fragment-based drug-discovery campaigns that are being used to probe their potential druggability.

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