Rajasekhar Neeli

The University of Manchester, Manchester, England, United Kingdom

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Publications (11)40.94 Total impact

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    ABSTRACT: We report the crystal structure of the FAD/NADPH-binding domain (FAD domain) of the biotechnologically important Bacillus megaterium flavocytochrome P450 BM3, the last domain of the enzyme to be structurally resolved. The structure was solved in both the absence and presence of the ligand NADP(+), identifying important protein interactions with the NADPH 2'-phosphate that helps to dictate specificity for NADPH over NADH, and involving residues Tyr974, Arg966, Lys972 and Ser965. The Trp1046 side chain shields the FAD isoalloxazine ring from NADPH, and motion of this residue is required to enable NADPH-dependent FAD reduction. Multiple binding interactions stabilize the FAD cofactor, including aromatic stacking with the adenine group from the side chains of Tyr860 and Trp854, and several interactions with FAD pyrophosphate oxygens, including bonding to tyrosines 828, 829 and 860. Mutagenesis of C773 and C999 to alanine was required for successful crystallization, with C773A predicted to disfavour intramolecular and intermolecular disulfide bonding. Multiangle laser light scattering analysis showed wild-type FAD domain to be near-exclusively dimeric, with dimer disruption achieved on treatment with the reducing agent dithiothreitol. By contrast, light scattering showed that the C773A/C999A FAD domain was monomeric. The C773A/C999A FAD domain structure confirms that Ala773 is surface exposed and in close proximity to Cys810, with this region of the enzyme's connecting domain (that links the FAD domain to the FMN-binding domain in P450 BM3) located at a crystal contact interface between FAD domains. The FAD domain crystal structure enables molecular modelling of its interactions with its cognate FMN (flavodoxin-like) domain within the BM3 reductase module.
    FEBS Journal 02/2012; 279(9):1694-706. · 4.25 Impact Factor
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    ABSTRACT: Bacillus megaterium P450 BM3 (BM3) is a P450/P450 reductase fusion enzyme, where the dimer is considered the active form in NADPH-dependent fatty acid hydroxylation. The BM3 W1046A mutant was generated, removing an aromatic "shield" from its FAD isoalloxazine ring. W1046A BM3 is a catalytically active NADH-dependent lauric acid hydroxylase, with product formation slightly superior to the NADPH-driven enzyme. The W1046A BM3 K(m) for NADH is 20-fold lower than wild-type BM3, and catalytic efficiency of W1046A BM3 with NADH and NADPH are similar in lauric acid oxidation. Wild-type BM3 also catalyzes NADH-dependent lauric acid hydroxylation, but less efficiently than W1046A BM3. A hypothesis that W1046A BM3 is inactive [15] helped underpin a model of electron transfer from FAD in one BM3 monomer to FMN in the other in order to drive fatty acid hydroxylation in native BM3. Our data showing W1046A BM3 is a functional fatty acid hydroxylase are consistent instead with a BM3 catalytic model involving electron transfer within a reductase monomer, and from FMN of one monomer to heme of the other [12]. W1046A BM3 is an efficient NADH-utilizing fatty acid hydroxylase with potential biotechnological applications.
    Archives of Biochemistry and Biophysics 03/2011; 507(1):75-85. · 3.37 Impact Factor
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    ABSTRACT: Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.
    Journal of Biological Chemistry 10/2008; 283(48):33406-16. · 4.65 Impact Factor
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    ABSTRACT: 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 Arg(199) and Arg(200) 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 K(m) values), but lower NADH K(m) values. Despite a lowered k(cat), the R199A/R200A mutant exhibited a 200-fold coenzyme specificity switch towards NADH, although substrate inhibition was observed at high NADH concentrations (K(i)=250 microM). Stopped-flow FAD reduction studies confirmed substantially increased NADPH K(d) 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 FADH(2)-NADP+ charge-transfer intermediate) for R200A FprA, albeit more slowly (k(lim)=58.1 s(-1) compared with >300 s(-1)) than in WT. All mutations caused positive shifts in FAD potential (approximately 40-65 mV). Binding of an NADPH analogue (tetrahydro-NADP) induced negative shifts in potential ( approximately 30-40 mV) only for variants with the R200A mutation, indicating distinctive effects of Arg(199)/Arg(200) 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.
    Biochemical Journal 09/2008; 417(1):103-12. · 4.65 Impact Factor
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    ABSTRACT: 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. Trp(359), 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 Trp(359) 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 Trp(359). 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 k(cat) and NAD(P)H K(m) values, but this led to improvements in catalytic efficiency (k(cat)/K(m)) with NADH as reducing coenzyme (9.6/18.8 muM(-1).min(-1) respectively, compared with 5.7 muM(-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 Trp(359) for flavin stability. The results demonstrate that the conserved Trp(359) is critical in regulating FprA FAD binding, thermodynamic properties, catalytic efficiency and coenzyme selectivity.
    Biochemical Journal 06/2008; 411(3):563-70. · 4.65 Impact Factor
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    ABSTRACT: The human pathogen Mycobacterium tuberculosis has made a dramatic resurgence in recent years. Drug resistant and multidrug resistant strains are prevalent, and novel antibiotic strategies are desperately needed to counter Mtb's global spread. The M. tuberculosis genome sequence revealed an unexpectedly high number of cytochrome P450 (P450) enzymes (20), and parallel studies indicated that P450-inhibiting azole drugs had potent anti-mycobacterial activity. This article reviews current knowledge of structure/function of P450s and redox partner systems in M. tuberculosis. Recent research has highlighted potential drug target Mtb P450s and provided evidence for roles of selected P450 isoforms in host lipid and sterol/steroid transformations. Structural analysis of key Mtb P450s has provided fundamental information on the nature of the heme binding site, P450 interactions with azole drugs, the biochemical nature of cytochrome P420, and novel mutational adaptations by which azole binding to P450s may be diminished to facilitate azole resistance.
    Archives of Biochemistry and Biophysics 09/2007; 464(2):228-40. · 3.37 Impact Factor
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    ABSTRACT: Flavocytochrome P450 (cytochrome P450) BM3 is an intensively studied model system within the P450 enzyme superfamily, and is a natural fusion of a P450 to its P450 reductase redox partner. The fusion arrangement enables efficient electron transfer within the enzyme and a catalytic efficiency that cannot be matched in P450 systems from higher organisms. P450 BM3's potential for industrially relevant chemical transformations is now recognized, and variants with biotechnological applications have been constructed. Simultaneously, structural and mechanistic studies continue to reveal the intricate mechanistic details of this enzyme, including its dimeric organization and the relevance of this quaternary structure to catalysis. Homologues of BM3 have been found in several bacteria and fungi, indicating important physiological functions in these microbes and enabling first insights into evolution of the enzyme family. This short paper deals with recent developments in our understanding of structure, function, evolution and biotechnological applications of this important P450 system.
    Biochemical Society Transactions 01/2007; 34(Pt 6):1173-7. · 2.59 Impact Factor
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    ABSTRACT: An extraordinary array of P450 (cytochrome P450) enzymes are encoded on the genome of the human pathogen Mycobacterium tuberculosis (Mtb) and in related mycobacteria and actinobacteria. These include the first characterized sterol 14alpha-demethylase P450 (CYP51), a known target for azole and triazole drugs in yeasts and fungi. To date, only two Mtb P450s have been characterized in detail: CYP51 and CYP121. The CYP121 P450 shows structural relationships with P450 enzymes involved in synthesis of polyketide antibiotics. Both P450s exhibit tight binding to a range of azole drugs (e.g. clotrimazole and fluconazole) and the same drugs also have potent effects on growth of mycobacteria (but not of e.g. Escherichia coli). Atomic structures are available for both Mtb CYP51 and CYP121, revealing modes of azole binding and intriguing mechanistic and structural aspects. This paper reviews our current knowledge of these and the other P450 systems in Mtb including recent data relating to the reversible conversion of the CYP51 enzyme between P450 (thiolate-co-ordinated) and P420 (thiol-co-ordinated) species on reduction of the haem iron in the absence of a P450 substrate. The accessory flavoprotein and iron-sulfur proteins required to drive P450 catalysis are also discussed, providing an overview of the current state of knowledge of Mtb P450 redox systems.
    Biochemical Society Transactions 01/2007; 34(Pt 6):1178-82. · 2.59 Impact Factor
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    ABSTRACT: In the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN(1)-to-heme(2)) electron transfer supports oxygenase activity in the BM3 dimer.
    FEBS Letters 11/2005; 579(25):5582-8. · 3.58 Impact Factor
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    ABSTRACT: Since its discovery in the 1980s, the fatty acid hydroxylase flavocytochrome P450 (cytochrome P450) BM3 (CYP102A1) from Bacillus megaterium has been adopted as a paradigm for the understanding of structure and mechanism in the P450 superfamily of enzymes. P450 BM3 was the first P450 discovered as a fusion to its redox partner--a eukaryotic-like diflavin reductase. This fact fuelled the interest in soluble P450 BM3 as a model for the mammalian hepatic P450 enzymes, which operate a similar electron transport chain using separate, membrane-embedded P450 and reductase enzymes. Structures of each of the component domains of P450 BM3 have now been resolved and detailed protein engineering and molecular enzymology studies have established roles for several amino acids in, e.g. substrate binding, coenzyme selectivity and catalysis. The potential of P450 BM3 for biotechnological applications has also been recognized, with variants capable of industrially important transformations generated using rational mutagenesis and forced evolution techniques. This paper focuses on recent developments in our understanding of structure and mechanism of this important enzyme and highlights important problems still to be resolved.
    Biochemical Society Transactions 09/2005; 33(Pt 4):747-53. · 2.59 Impact Factor
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    ABSTRACT: Flavocytochrome P450 BM3 is a member of the diflavin reductase enzyme family. Members include cytochrome P450 reductase, nitric-oxide synthase, methionine synthase reductase, and novel oxidoreductase 1. These enzymes show a strong preference for NADPH over NADH as reducing coenzyme. An aromatic residue stacks over the FAD isoalloxazine ring in each enzyme, and in some cases it is important in controlling coenzyme specificity. In P450 BM3, the aromatic residue inferred from sequence alignments to stack over the FAD is Trp-1046. Mutation to Ala-1046 and His-1046 effected a remarkable coenzyme specificity switch. P450 BM3 W1046A/W106H FAD and reductase domains are efficient NADH-dependent ferricyanide reductases with selectivity coefficients (k(cat)/K(m)(NADPH)/k(cat)/K(m)(NADH)) of 1.5, 67, and 8571 for the W1046A, W1046H, and wild-type reductase domains, respectively. Stopped-flow photodiode array absorption studies indicated a charge-transfer intermediate accumulated in the W1046A FAD domain (and to a lesser extent in the W1046H FAD domain) and was attributed to formation of a reduced FADH(2)-NAD(P)(+) charge-transfer species, suggesting a relatively slow rate of release of NAD(P)(+) from reduced enzymes. Unlike wild-type enzymes, there was no formation of the blue semiquinone species observed during reductive titration of the W0146A/W146H FAD and reductase domains with dithionite or NAD(P)H. This was a consequence of elevation of the semiquinone/hydroquinone couple of the FAD with respect to the oxidized/semiquinone couple, and a concomitant approximately 100-mV elevation in the 2-electron redox couple for the enzyme-bound FAD (-320, -220, and -224 mV in the wild-type, W1046A, and W1046H FAD domains, respectively).
    Journal of Biological Chemistry 06/2005; 280(18):17634-44. · 4.65 Impact Factor

Publication Stats

185 Citations
40.94 Total Impact Points

Institutions

  • 2005–2012
    • The University of Manchester
      • • Faculty of Life Sciences
      • • School of Chemical Engineering and Analytical Science
      Manchester, England, United Kingdom
  • 2005–2007
    • University of Leicester
      • Department of Biochemistry
      Leicester, ENG, United Kingdom