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N S Scrutton

Publications and source records attributed to N S Scrutton.

At least 19 recordsLinked to original sources
PubMed2006

Flavocytochrome P450 BM3 and the origin of CYP102 fusion species.

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.

Bacterial Proteins↗
PubMed2005

Flavocytochrome P450 BM3: an update on structure and mechanism of a biotechnologically important enzyme.

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.

Animals↗
PubMed2005

Flavoenzyme catalysed oxidation of amines: roles for flavin and protein-based radicals.

Amines are a carbon source for the growth of a number of bacterial species and they also play key roles in neurotransmission, cell growth and differentiation, and neoplastic cell proliferation. Enzymes have evolved to catalyse these reactions and these oxidoreductases can be grouped into the flavoprotein and quinoprotein families. The mechanism of amine oxidation catalysed by the quinoprotein amine oxidases is understood reasonably well and occurs through the formation of enzyme-substrate covalent adducts with TPQ (topaquinone), TTQ (tryptophan tryptophylquinone), CTQ (cysteine tryptophylquinone) and LTQ (lysine tyrosyl quinone) redox centres. Oxidation of amines by flavoenzymes is less well understood. The role of protein-based radicals and flavin semiquinone radicals in the oxidation of amines is discussed.

Amines↗
PubMed2005

Crystal structure of DMGO provides a prototype for a new tetrahydrofolate-binding fold.

The crystal structure of DMGO (dimethylglycine oxidase) from Arthrobacter globiformis in complex with folate compounds has revealed a novel THF (tetrahydrofolate)-binding fold [Leys, Basran and Scrutton (2003) EMBO J. 22, 4038-4048]. This fold is widespread among folate-binding proteins. The crystal structures of aminomethyltransferase (T-protein), YgfZ and TrmE all reveal similar THF-binding folds despite little similarity in sequence or function. The THF-binding site is highly specific for reduced folate compounds and most members of this fold family enhance the nucleophilic character of the THF N10 position.

Binding Sites↗
PubMed2005

Biodiversity of cytochrome P450 redox systems.

P450s (cytochrome P450 mono-oxygenases) are a superfamily of haem-containing mono-oxygenase enzymes that participate in a wide range of biochemical pathways in different organisms from all of the domains of life. To facilitate their activity, P450s require sequential delivery of two electrons passed from one or more redox partner enzymes. Although the P450 enzymes themselves show remarkable similarity in overall structure, it is increasingly apparent that there is enormous diversity in the redox partner systems that drive the P450 enzymes. This paper examines some of the recent advances in our understanding of the biodiversity of the P450 redox apparatus, with a particular emphasis on the redox systems in the pathogen Mycobacterium tuberculosis.

Bacterial Proteins↗
PubMed2003

Electron transfer in human cytochrome P450 reductase.

Cytochrome P450 reductase (CPR) is a diflavin enzyme responsible for electron donation to mammalian cytochrome P450 enzymes in the endoplasmic reticulum. Dissection of the enzyme into functional domains and studies by site-directed mutagenesis have enabled detailed characterization of the mechanism of electron transfer using stopped-flow and equilibrium-perturbation methods, and redox potentiometry. These studies and the mechanism of electron transfer in CPR are reported herein.

Amino Acid Sequence↗
PubMed2003

Cytochromes P450: novel drug targets in the war against multidrug-resistant Mycobacterium tuberculosis.

Novel drug strategies are desperately needed to combat the global threat posed by multidrug-resistant strains of Mycobacterium tuberculosis (Mtb). The genome sequence of Mtb has revealed an unprecedented number of cytochrome P450 enzymes in a prokaryote, suggesting fundamental physiological roles for many of these enzymes. Several azole drugs (known inhibitors of cytochromes P450) have been shown to have potent anti-mycobacterial activity, and the most effective azoles have extremely tight binding constants for one of the Mtb P450s (CYP121). The structure of CYP121 has been determined at atomic resolution, revealing novel features of P450 structure, including mixed haem conformations and putative proton-relay pathways from protein surface to haem iron. The structure provides both a platform for investigation of structure/mechanism of cytochrome P450, and for design of inhibitor molecules as novel anti-tubercular agents.

Antitubercular Agents↗
PubMed2001

Effects of environment on flavin reactivity in morphinone reductase: analysis of enzymes displaying differential charge near the N-1 atom and C-2 carbonyl region of the active-site flavin.

The side chain of residue Arg(238) in morphinone reductase (MR) is located close to the N-1/C-2 carbonyl region of the flavin isoalloxazine ring. During enzyme reduction negative charge develops in this region of the flavin. The positioning of a positively charged side chain in the N-1/C-2 carbonyl region of protein-bound flavin is common to many flavoprotein enzymes. To assess the contribution made by Arg(238) in stabilizing the reduced flavin in MR we isolated three mutant forms of the enzyme in which the position of the positively charged side chain was retracted from the N-1/C-2 carbonyl region (Arg(238)-->Lys), the positive charge was removed (Arg(238)-->Met) or the charge was reversed (Arg(238)-->Glu). Each mutant enzyme retains flavin in its active site. Potentiometric studies of the flavin in the wild-type and mutant forms of MR indicate that the flavin semiquinone is not populated to any appreciable extent. Reduction of the flavin in each enzyme is best described by a single Nernst function, and the values of the midpoint reduction potentials (E(12)) for each enzyme fall within the region of -247+/-10 mV. Stopped-flow studies of NADH binding to wild-type and mutant MR enzymes reveal differences in the kinetics of formation and decay of an enzyme-NADH charge-transfer complex, reflecting small perturbations in active-site geometry. Reduced rates of hydride transfer in the mutant enzymes are attributed to altered geometrical alignment of the nicotinamide coenzyme with FMN rather than major perturbations in reduction potential, and this is supported by an observed entropy-enthalpy compensation effect on the hydride transfer reaction throughout the series of enzymes. The data indicate, in contrast with dogma, that the presence of a positively charged side chain close to the N-1/C-2 carbonyl region of the flavin in MR is not required to stabilize the reduced flavin. This finding may have general implications for flavoenzyme catalysis, since it has generally been assumed that positive charge in this region has a stabilizing effect on the reduced form of flavin.

Bacterial Proteins↗
PubMed2001

Optimizing the Michaelis complex of trimethylamine dehydrogenase: identification of interactions that perturb the ionization of substrate and facilitate catalysis with trimethylamine base.

Recent evidence from isotope studies supports the view that catalysis by trimethylamine dehydrogenase (TMADH) proceeds from a Michaelis complex involving trimethylamine base and not, as thought previously, trimethylammonium cation. In native TMADH reduction of the flavin by substrate (perdeuterated trimethylamine) is influenced by two ionizations in the Michaelis complex with pK(a) values of 6.5 and 8.4; maximal activity is realized in the alkaline region. The latter ionization has been attributed to residue His-172 and, more recently, the former to the ionization of substrate itself. In the Michaelis complex, the ionization of substrate (pK(a) approximately 6.5 for perdeuterated substrate) is perturbed by approximately -3.3 to -3.6 pH units compared with that of free trimethylamine (pK(a) = 9.8) and free perdeuterated trimethylamine (pK(a) = 10.1), respectively, thus stabilizing trimethylamine base by approximately 2 kJ mol(-1). We show, by targeted mutagenesis and stopped-flow studies that this reduction of the pK(a) is a consequence of electronic interaction with residues Tyr-60 and His-172, thus these two residues are key for optimizing catalysis in the physiological pH range. We also show that residue Tyr-174, the remaining ionizable group in the active site that we have not targeted previously by mutagenesis, is not implicated in the pH dependence of flavin reduction. Formation of a Michaelis complex with trimethylamine base is consistent with a mechanism of amine oxidation that we advanced in our previous computational and kinetic studies which involves nucleophilic attack by the substrate nitrogen atom on the electrophilic C4a atom of the flavin isoalloxazine ring. Stabilization of trimethylamine base in the Michaelis complex over that in free solution is key to optimizing catalysis at physiological pH in TMADH, and may be of general importance in the mechanism of other amine dehydrogenases that require the unprotonated form of the substrate for catalysis.

Binding Sites↗
PubMed2001

Kinetics of CO binding to the haem domain of murine inducible nitric oxide synthase: differential effects of haem domain ligands.

The binding of CO to the murine inducible nitric oxide synthase (iNOS) oxygenase domain has been studied by laser flash photolysis. The effect of the (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)) cofactor L-arginine and several Type I L-arginine analogues/ligands on the rates of CO rebinding has been evaluated. The presence of BH(4) in the iNOS active site has little effect on the rebinding of protein-caged haem-CO pairs (geminate recombination), but decreases the bimolecular association rates 2-fold. Addition of L-arginine to the BH(4)-bound complex completely abolishes geminate recombination and results in a further 80-fold decrease in the overall rate of bimolecular association. Three of the Type I ligands, S-ethylisothiourea, L-canavanine and 2,5-lutidine, displaced the CO from the haem iron upon addition to the iNOS oxygenase domain. The Type I ligands significantly decreased the rate of bimolecular binding of CO to the haem iron after photolysis. Most of these ligands also completely abolished geminate recombination. These results are consistent with a relatively open distal pocket that allows CO to bind unhindered in the active site of murine iNOS in the absence of L-arginine or BH(4). In the presence of BH(4) and L-arginine, however, the enzyme adopts a more closed structure that can greatly reduce ligand access to the haem iron. These observations are discussed in terms of the known structure of iNOS haem domain and solution studies of ligand binding in iNOS and neuronal NOS isoenzymes.

Animals↗
PubMed2001

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.

Androstadienes↗
PubMed2001

Protein dynamics enhance electronic coupling in electron transfer complexes.

Electron-transferring flavoproteins (ETFs) from human and Paracoccus denitrificans have been analyzed by small angle x-ray scattering, showing that neither molecule exists in a rigid conformation in solution. Both ETFs sample a range of conformations corresponding to a large rotation of domain II with respect to domains I and III. A model of the human ETF.medium chain acyl-CoA dehydrogenase complex, consistent with x-ray scattering data, indicates that optimal electron transfer requires domain II of ETF to rotate by approximately 30 to 50 degrees toward domain I relative to its position in the x-ray structure. Domain motion establishes a new "robust engineering principle" for electron transfer complexes, tolerating multiple configurations of the complex while retaining efficient electron transfer.

Electron Transport↗
PubMed2001

Deuterium isotope effects during carbon-hydrogen bond cleavage by trimethylamine dehydrogenase. Implications for mechanism and vibrationally assisted hydrogen tunneling in wild-type and mutant enzymes.

His-172 and Tyr-169 are components of a triad in the active site of trimethylamine dehydrogenase (TMADH) comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies with trimethylamine as substrate have indicated that mutation of His-172 to Gln reduces the limiting rate constant for flavin reduction approximately 10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton, N. S. (1999) Biochem. J. 341, 307-314). A kinetic isotope effect (KIE = k(H)/k(D)) accompanies flavin reduction by H172Q TMADH, the magnitude of which varies significantly with solution pH. With trimethylamine, flavin reduction by H172Q TMADH is controlled by a single macroscopic ionization (pK(a) = 6.8 +/- 0.1). This ionization is perturbed (pK(a) = 7.4 +/- 0.1) in reactions with perdeuterated trimethylamine and is responsible for the apparent variation in the KIE with solution pH. At pH 9.5, where the functional group controlling flavin reduction is fully ionized, the KIE is independent of temperature in the range 277-297 K, consistent with vibrationally assisted hydrogen tunneling during breakage of the substrate C-H bond. Y169F TMADH is approximately 4-fold more compromised than H172Q TMADH for hydrogen transfer, which occurs non-classically. Studies with Y169F TMADH suggest partial thermal excitation of substrate prior to hydrogen tunneling by a vibrationally assisted mechanism. Our studies illustrate the varied effects of compromising mutations on tunneling regimes in enzyme molecules.

Aspartic Acid↗
PubMed2001

alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone.

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is +153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) < -250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.

Arginine↗
PubMed2001

Kinetics of CO binding and CO photodissociation in Pseudomonas stutzeri cd(1) nitrite reductase: probing the role of extended N-termini in fast structural relaxation upon CO photodissociation.

cd(1) nitrite reductase from Pseudomonas stutzeri is a di-haem- containing enzyme, comprising a c-type haem and a d-type haem. Studies with the highly related cd(1) nitrite reductase of Pseudomonas aeruginosa have established that this enzyme undergoes fast (microsecond) and global structural relaxation upon CO photodissociation from the reduced enzyme. A key difference between the Ps. aeruginosa and Ps. stutzeri enzyme is the absence of a flexible N-terminal extension in the Ps. stutzeri enzyme. In Ps. aeruginosa cd(1) nitrite reductase the N-terminal extension wraps around the second subunit of the homodimer and with Tyr(10) stabilizing a water molecule co-ordinated to the d(1)-haem. Given the intimate association of the N-terminal extension with the d(1)-haem, we hypothesized that the presence of the N-terminal extension likely contributes to the fast structural reorganization seen during photodissociation of CO from the reduced enzyme. In the present study we have investigated the kinetics of CO association and CO photodissociation of Ps. stutzeri cd(1) nitrite reductase (which lacks the N-terminal arm seen in the Ps. aeruginosa enzyme) to probe the role and influence of the N-terminal arm in the fast global structural reorganization seen with Ps. aeruginosa. Surprisingly, we find that Ps. stutzeri cd(1) nitrite reductase also undergoes fast structural reorganization during CO photodissociation. We also show, in stopped-flow experiments, that the kinetics of CO binding and dissociation with reduced Ps. stutzeri cd(1) nitrite reductase are similar to those observed with Ps. aeruginosa enzyme, thus ruling out a major role for the N-terminal flexible arm found in Ps. aeruginosa in the kinetics of these processes. Our data indicate that global structural reorganization following CO photodissociation is an intrinsic property of the haem domains in cd(1) nitrite reductases. The absence of an N-terminal extension, as in the Ps. stutzeri cd(1) nitrite reductase, does not lead to loss of global structural reorganization following CO photodissociation.

Carbon Monoxide↗
PubMed2001

Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains.

The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (>500 s(-)(1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization ( approximately 200 s(-)(1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (approximately 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH. The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].

Animals↗
PubMed2001

Two-iron rubredoxin of Pseudomonas oleovorans: production, stability and characterization of the individual iron-binding domains by optical, CD and NMR spectroscopies.

A minigene encoding the C-terminal domain of the 2Fe rubredoxin of Pseudomonas oleovorans was created from the parental alk G gene contained in the expression plasmid pKK223-3. The vector directed the high-level production of the C-terminal domain of this rubredoxin; a simple procedure was used to purify the recombinant domain in the 1Fe form. The 1Fe form of the C-terminal domain was readily converted into the apoprotein and cadmium forms after precipitation with trichloroacetic acid and resolubilization in the presence or absence of cadmium chloride respectively. In steady-state assays, the recombinant 1Fe C-terminal domain is redox-active and able to transfer electrons from reduced rubredoxin reductase to cytochrome c. The absorption spectrum and dichroic features of the CD spectrum for the iron- and cadmium-substituted C-terminal domain are similar to those reported for the iron- and cadmium-substituted Desulfovibrio gigas rubredoxin [Henehen, Pountney, Zerbe and Vasak (1993) Protein Sci. 2, 1756-1764]. Difference absorption spectroscopy of the cadmium-substituted C-terminal domain revealed the presence of four Gaussian-resolved maxima at 202, 225, 240 and 276 nm; from Jørgensen's electronegativity theory, the 240 nm band is attributable to a CysS-Cd(II) charge-transfer excitation. Attempts to express the N-terminal domain of the 2Fe rubredoxin directly from a minigene were unsuccessful. However, the N-terminal domain was isolated through cleavage of an engineered 2Fe rubredoxin in which a factor Xa proteolysis site had been introduced into the putative interdomain linker. The N-terminal domain is characterized by absorption spectra typical of the 1Fe rubredoxins. The domain is folded as determined by CD and NMR spectroscopies and is redox-active. However, the N-terminal domain is less stable than the isolated C-terminal domain, a finding consistent with the known properties of the full-length 2Fe and cadmium-substituted Ps. oleovorans rubredoxin.

Amino Acid Sequence↗