Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b(5) reductase

Cytochrome b(5) reductase (cb5r) catalyzes the transfer of reducing equivalents from NADH to cytochrome b(5). Utilizing an efficient heterologous expression system that produces a histidine-tagged form of the hydrophilic, diaphorase domain of the enzyme, site-directed mutagenesis has been used to generate cb5r mutants with substitutions at position 91 in the primary sequence. Arginine 91 is an important residue in binding the FAD prosthetic group and part of a conserved "RxY(T)(S)xx(S)(N)" sequence motif that is omnipresent in the "ferredoxin:NADP(+) reductase" family of flavoproteins. Arginine 91 was replaced with K, L, A, P, D, Q, and H residues, respectively, and all the mutant proteins purified to homogeneity. Individual mutants were expressed with variable efficiency and all exhibited molecular masses of approximately 32 kDa. With the exception of R91H, all the mutants retained visible absorption spectra typical of a flavoprotein, the former being produced as an apoprotein. Visible absorption spectra of R91A, L, and P were red shifted with maxima at 458 nm, while CD spectra indicated an altered FAD environment for all the mutants except R91K. Fluorescence spectra showed a reduced degree of intrinsic flavin fluorescence quenching for the R91K, A, and P, mutants, while thermal stability studies suggested all the mutants, except R91K, were somewhat less stable than the wild-type domain. Initial-rate kinetic measurements demonstrated that the mutants exhibited decreased NADH:ferricyanide reductase activity with the R91P mutant retaining the lowest activity, corresponding to a k(cat) of 283 s(-1) and a K(NADH)(m) of 105 microM, when compared to the wild-type domain (k(cat) = 800 s(-1) K(NADH)(m) = 6 microM). These results demonstrate that R91 is not essential for FAD binding in cb5r; however, mutation of R91 perturbs the flavin environment and alters both diaphorase substrate recognition and utilization.     

Kinetic constants of signal peptidase I using cytochrome b5 as a precursor substrate.

A procedure is described for measuring Escherichia coli signal peptidase I activity which exploits an intact precursor protein composed of the alkaline phosphatase signal peptide fused to the full length mammalian cytochrome b5. This cytochrome b5 precursor protein has been extensively characterised and shown to be processed accurately by purified signal peptidase I [Protein Expr. Purif. 7 (1996) 237]. The amphipathic, chimaeric cytochrome b5 precursor was isolated in mg quantities in a highly homogeneous state under non-denaturing conditions. The processing of the cytochrome b5 precursor by signal peptidase displayed Michaelis-Menten kinetics with K(m)=50 microM and k(cat)=11 s(-1). The K(m) was 20-fold lower than that obtained with signal peptide substrates and 3-fold higher than that reported for pro-OmpA-nuclease A precursor fusion. The corresponding turnover number, k(cat), was four orders of magnitude greater than the peptide substrates but was 2-fold lower than pro-OmpA-nuclease A precursor fusion. These results confirm that both the affinities and the catalytic power of the signal peptidase are significantly higher for macromolecular precursor substrates than for the shorter signal peptide substrates.     

Effects of flavin-binding motif amino acid mutations in the NADH-cytochrome b5 reductase catalytic domain on protein stability and catalysis

Porcine NADH-cytochrome b5 reductase catalytic domain (Pb5R) has the RXY(T/S)+(T/S) flavin-binding motif that is highly conserved among the structurally related family of flavoprotein reductases. Mutations were introduced that alter the Arg(63), Tyr(65), and Ser(99) residues within this motif. The mutation of Tyr(65) to either alanine or phenylalanine destabilized the protein, produced an accelerated release of FAD in the presence of 1.5 M guanidine hydrochloride, and decreased the k(cat) values of the enzyme. These results indicate that Tyr(65) contributes to the stability of the protein and is important in the electron transfer from NADH to FAD. The mutation of Ser(99) to either alanine or valine, and of Arg(63) to either alanine or glutamine increased both the K(m) values for NADH (K(m)(NADH)) and the dissociation constant for NAD(+) (K(d)(NAD+)). However, the mutation of Ser(99) to threonine and of Arg(63) to lysine had very little effect on the K(m)(NADH) and K(d)(NAD+) values, and resulted in small changes in the absorption and circular dichroism spectra. These results suggest that the hydroxyl group of Ser(99) and the positive charge of Arg(63) contribute to the maintenance of the properties of FAD and to the effective binding of Pb5R to both NADH and NAD(+). In addition, the mutation of Arg(63) to either alanine or glutamine increased the apparent K(m) values for porcine cytochrome b5 (Pb5), while changing Arg(63) to lysine did not. The positive charge of Arg(63) may regulate the electron transfer through the electrostatic interaction with Pb5. These results substantiate the important role of the flavin-binding motif in Pb5R.     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

Kinetic and mechanistic properties of biotin sulfoxide reductase

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase catalyzes the reduction of d-biotin d-sulfoxide (BSO) to biotin. Initial rate studies of the homogeneous recombinant enzyme, expressed in Escherichia coli, have demonstrated that the purified protein utilizes NADPH as a facile electron donor in the absence of any additional auxiliary proteins. We have previously shown [Pollock, V. V., and Barber, M. J. (1997) J. Biol. Chem. 272, 3355-3362] that, at pH 8 and in the presence of saturating concentrations of BSO, the enzyme exhibits, a marked preference for NADPH (k(cat,app) = 500 s(-1), K(m,app) = 269 microM, and k(cat,app)/K(m,app) = 1.86 x 10(6) M(-1) s(-1)) compared to NADH (k(cat,app) = 47 s(-1), K(m,app) = 394 microM, and k(cat,app)/K(m,app) = 1.19 x 10(5) M(-1) s(-1)). Production of biotin using NADPH as the electron donor was confirmed by both the disk biological assay and by reversed-phase HPLC analysis of the reaction products. The purified enzyme also utilized ferricyanide as an artificial electron acceptor, which effectively suppressed biotin sulfoxide reduction and biotin formation. Analysis of the enzyme isolated from tungsten-grown cells yielded decreased reduced methyl viologen:BSO reductase, NADPH:BSO reductase, and NADPH:FR activities, confirming that Mo is required for all activities. Kinetic analyses of substrate inhibition profiles revealed that the enzyme followed a Ping Pong Bi-Bi mechanism with both NADPH and BSO exhibiting double competitive substrate inhibition. Replots of the 1/v-axes intercepts of the parallel asymptotes obtained at several low concentrations of fixed substrate yielded a K(m) for BSO of 714 and 65 microM for NADPH. In contrast, utilizing NADH as an electron donor, the replots yielded a K(m) for BSO of 132 microM and 1.25 mM for NADH. Slope replots of data obtained at high concentrations of BSO yielded a K(i) for BSO of 6.10 mM and 900 microM for NADPH. Kinetic isotope studies utilizing stereospecifically deuterated NADPD indicated that BSO reductase uses specifically the 4R-hydrogen of the nicotinamide ring. Cyanide inhibited NADPH:BSO and NADPH:FR activities in a reversible manner while diethylpyrocarbonate treatment resulted in complete irreversible inactivation of the enzyme concomitant with molybdenum cofactor release, indicating that histidine residues are involved in cofactor-binding.     

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.     

Heterologous expression of enzymopenic methemoglobinemia variants using a novel NADH:cytochrome c reductase fusion protein

Hereditary enzymopenic methemoglobinemia is a rare disease that predominantly results from defects in either the erythrocytic (type I) or microsomal (type II) forms of the enzyme NADH:cytochrome b5 reductase (EC 1.6.2.2). All 25 currently identified type I and type II methemoglobinemia mutants have been expressed in Escherichia coli using a novel six histidine-tagged rat cytochrome b5/cytochrome b5 reductase fusion protein designated NADH:cytochrome c reductase (H6NCR). All 25 H6NCR variants were isolated and demonstrated to result in two groups of expression products. The first group of 16 mutants, which included the majority of the type I mutants, included K116Q, P131L, L139P, T183S, M193V, S194P, P211L, L215P, A245T, A245V, C270Y, E279K, V305R, V319M, M340-, and F365-, and yielded full-length fusion proteins that retained variable levels of NADH:cytochrome c reductase (NADH:CR) activity, ranging from approximately 2% (M340-) to 92% (K116Q) of that of the wild-type fusion protein. In contrast, the remaining nine mutants that represented the majority of the type II variants, comprised a second group that included Y109*, R124Q, Q143*, R150*, P162H, V172M, R226*, C270R, and R285*, and resulted in truncated H6NCR variants that retained the amino-terminal cytochrome b5 domain but were devoid of NADH:CR activity due to the absence of the cytochrome b5 reductase flavin domain. Kinetic analyses of the first group of full-length mutant fusion proteins indicated that values for both kcat and Km(NADH) were decreased and increased, respectively, indicating that the various mutations affected both substrate affinity and/or turnover. However, for the second group, the truncated products were the result of incomplete production of the carboxyl-terminal flavin-containing domain or instability of the expression products due to improper folding and/or lack of flavin incorporation.     

Quaternary structure and composition of squash NADH:nitrate reductase

NADH:nitrate reductase (EC 1.6.6.1) was isolated from squash cotyledons (Cucurbita maxima L.) by a combination of Blue Sepharose and zinc-chelate affinity chromatographies followed by gel filtration on Bio-Gel A-1.5m. These preparations gave a single protein staining band (Mr = 115,000) on sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is homogeneous. The native Mr of nitrate reductase was found to be 230,000, with a minor form of Mr = 420,000 also occurring. These results indicate that the native nitrate reductase is a homodimer of Mr = 115,000 subunits. Acidic amino acids predominate over basic amino acids, as shown both by the amino acid composition of the enzyme and an isoelectric point for nitrate reductase of 5.7. The homogeneous nitrate reductase had a UV/visible spectrum typical of a b-type cytochrome. The enzyme was found to contain one each of flavin (as FAD), heme iron, molybdenum, and Mo-pterin/Mr = 115,000 subunit. A model is proposed for squash nitrate reductase in which two Mr = 115,000 subunits are joined to made the native enzyme. Each subunit contains 1 eq of FAD, cytochrome b, and molybdenum/Mo-pterin.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

Modification of the nucleotide cofactor-binding site of cytochrome P-450 reductase to enhance turnover with NADH in Vivo

NADPH-cytochrome P-450 reductase is the electron transfer partner for the cytochromes P-450, heme oxygenase, and squalene monooxygenase and is a component of the nitric-oxide synthases and methionine-synthase reductase. P-450 reductase shows very high selectivity for NADPH and uses NADH only poorly. Substitution of tryptophan 677 with alanine has been shown to yield a 3-fold increase in turnover with NADH, but profound inhibition by NADP(+) makes the enzyme unsuitable for in vivo applications. In the present study site-directed mutagenesis of amino acids in the 2'-phosphate-binding site of the NADPH domain, coupled with the W677A substitution, was used to generate a reductase that was able to use NADH efficiently without inhibition by NADP(+). Of 11 single, double, and triple mutant proteins, two (R597M/W677A and R597M/K602W/W677A) showed up to a 500-fold increase in catalytic efficiency (k(cat)/K(m)) with NADH. Inhibition by NADP(+) was reduced by up to 4 orders of magnitude relative to the W677A protein and was equal to or less than that of the wild-type reductase. Both proteins were 2-3-fold more active than wild-type reductase with NADH in reconstitution assays with cytochrome P-450 1A2 and with squalene monooxygenase. In a recombinant cytochrome P-450 2E1 Ames bacterial mutagenicity assay, the R597M/W677A protein increased the sensitivity to dimethylnitrosamine by approximately 2-fold, suggesting that the ability to use NADH afforded a significant advantage in this in vivo assay.     

Engineering and characterization of a NADPH-utilizing cytochrome b5 reductase

Microsomal cytochrome b(5) reductase (EC 1.6.2.2) catalyzes the reduction of ferricytochrome b(5) using NADH as the physiological electron donor. Site-directed mutagenesis has been used to engineer the soluble rat cytochrome b(5) reductase diaphorase domain to utilize NADPH as the preferred electron donor. Single and double mutations at residues D239 and F251 were made in a recombinant expression system that corresponded to D239E, S and T, F251R, and Y, D239S/F251R, D239S/F251Y, and D239T/F251R, respectively. Steady-state turnover measurements indicated that D239S/F251Y was bispecific while D239T, D239S/F251R, and D239T/F251R were each NADPH-specific. Wild-type (WT) cytochrome b(5) reductase showed a 3700-fold preference for NADH whereas the mutant with the highest NADPH efficiency, D239T, showed an 11-fold preference for NADPH, a 39200-fold increase. Wild-type cytochrome b(5) reductase only formed a stable charge-transfer complex with NADH while D239T formed complexes with both NADH and NADPH. The rates of hydride ion transfer, determined by stopped-flow kinetics, were k(NADH-WT) = 130 s(-1), k(NADPH-WT) = 5 s(-1), k(NADH-D239T) = 180 s(-1), and k(NADPH-D239T) = 73 s(-1). K(s) determinations by differential spectroscopy demonstrated that D239T could bind nonreducing pyridine nucleotides with a phosphate or a hydroxyl substituent at the 2' position, whereas wild-type cytochrome b(5) reductase would only bind 2' hydroxylated molecules. Oxidation-reduction potentials (E degrees ', n = 2) for the flavin cofactor were WT = -268 mV, D239T = -272 mV, WT+NAD(+) = -190 mV, D239T+NAD(+) = -206 mV, WT+NADP(+) = -253 mV, and D239T+NADP(+) = -215 mV, which demonstrated the thermodynamic contribution of NADP(+) binding to D239T. The crystal structures of D239T and D239T in complex with NAD(+) indicated that the loss of the negative electrostatic surface that precluded 2' phosphate binding in the wild-type enzyme was primarily responsible for the observed improvement in the use of NADPH by the D239T mutant.     

Structure of a cDNA for Ciona Cytochrome b(5) and the ubiquitous expression of mRNA in embryonic tissues

A cDNA clone for cytochrome b(5) was isolated from a cDNA library of an ascidian, Ciona savignyi, by a plaque hybridization method using a digoxigenin-labeled cDNA for the soluble form of human cytochrome b(5). The cDNA is composed of 5'- and 3'-noncoding sequences, and a 396-base pair coding sequence. The 3'-noncoding sequence contains polyadenylation signal sequences. The amino acid sequence of 132 residues deduced from the nucleotide sequence of the cDNA showed 61% identity and 82% similarity to the cytochrome b(5) of another ascidian species, Polyandrocarpa misakiensis, which we previously cloned. The amino-terminal hydrophilic domain of 98 residues contains well-conserved structures around two histidine residues for heme binding. A cDNA expression system was constructed to prepare a putative soluble form of Ciona cytochrome b(5). The recombinant soluble cytochrome b(5) showed an asymmetrical absorption spectrum at 560 nm as is shown by mammalian cytochromes b(5) upon reduction with NADH and NADH-cytochrome b(5) reductase. The recombinant Ciona cytochrome b(5) is reduced by NADH-cytochrome b(5) reductase with an apparent K(m) value of 3.3 microM. This value is similar to that of the cytochrome b(5) of Polyandrocarpa misakiensis. The expression of Ciona cytochrome b(5) mRNA during development was examined by an in situ hybridization method and ubiquitous expression in embryonic tissues was observed. The results indicate that cytochrome b(5) plays important roles in various metabolic processes during development.     

Bacterial expression of the molybdenum domain of assimilatory nitrate reductase: production of both the functional molybdenum-containing domain and the nonfunctional tungsten analog

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex molybdenum-, cytochrome b(557)- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. To facilitate structure/function studies of the individual molybdenum center, we have developed bacterial expression systems for the heterologous production of the 541 residue amino-terminal, molybdenum center-containing domain of spinach nitrate reductase either as a six-histidine-tagged variant or as a glutathione-S-transferase-tagged fusion protein. Expression of the his-tagged molybdenum domain in Escherichia coli BL21(DE3) cells under anaerobic conditions yielded a 55-kDa domain with a specific activity of 1.5 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 8 mciroM. In contrast, expression of the molybdenum domain as a GST-tagged fusion protein in E. coli TP1000(MobA(-) strain) cells under aerobic conditions yielded an 85-kDa fusion protein with a specific activity of 10.8 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 12 microM. Fluorescence analysis indicated that both forms of the molybdenum domain contained the cofactor, MPT, although the MPT content was higher in the GST-fusion domain. Inductively coupled plasma mass spectrometric analysis of both the his-tagged and GST-fusion protein domain samples indicated Mo/protein ratios of 0.44 and 0.93, respectively, confirming a very high level of Mo incorporation in the GST-fusion protein. Expression of the GST-fusion protein in TP1000 cells in the presence of elevated tungsten concentrations resulted in an 85-kDa fusion protein that contained MPT but which was devoid of nitrate-reducing activity. Partial reduction of the molybdenum domain resulted in the generation of an axial Mo(V) EPR species with g values of 1.9952, 1.9693, and 1.9665, respectively, and exhibiting superhyperfine coupling to a single exchangeable proton, analogous to that previously observed for the native enzyme. In contrast, the tungsten-substituted MPT-containing domain yielded a W(V) EPR species with g values of 1.9560, 1.9474, and 1.9271, respectively, with unresolved superhyperfine interaction. NADH:nitrate reductase activity could be reconstituted using the GST-molybdenum domain fusion protein in the presence of the recombinant forms of the spinach nitrate reductase' flavin- and heme-containing domains.     

Vibrio harveyi NADPH-FMN oxidoreductase arg203 as a critical residue for NADPH recognition and binding

Luminous bacteria contain three types of NAD(P)H-FMN oxidoreductases (flavin reductases) with different pyridine nucleotide specificities. Among them, the NADPH-specific flavin reductase from Vibrio harveyi exhibits a uniquely high preference for NADPH. In comparing the substrate specificity, crystal structure, and primary sequence of this flavin reductase with other structurally related proteins, we hypothesize that the conserved Arg203 residue of this reductase is critical to the specific recognition of NADPH. The mutation of this residue to an alanine resulted in only small changes in the binding and reduction potential of the FMN cofactor, the K(m) for the FMN substrate, and the k(cat). In contrast, the K(m) for NADPH was increased 36-fold by such a mutation. The characteristic perturbation of the FMN cofactor absorption spectrum upon NADP(+) binding by the wild-type reductase was abolished by the same mutation. While the k(cat)/K(m,NADPH) was reduced from 1990 x 10(5) to 46 x 10(5) M(-1) min(-1) by the mutation, the mutated variant showed a k(cat)/K(m,NADH) of 4 x 10(5) M(-1) min(-1), closely resembling that of the wild-type reductase. The deuterium isotope effects (D)V and (D)(V/K) for (4R)-[4-(2)H]-NADPH were 1.7 and 1.4, respectively, for the wild-type reductase but were increased to 3.8 and 4.0, respectively, for the mutated variant. Such a finding indicates that the rates of NADPH and NADP(+) dissociation in relation to the isotope-sensitive redox steps were both increased as a result of the mutation. These results all provide support to the critical role of the Arg203 in the specific recognition and binding of NADPH.     

Characterisation of a bis(5'-nucleosyl)-tetraphosphatase (asymmetrical) from Drosophila melanogaster

The intracellular functions of diadenosine polyphosphates are still poorly defined. To understand these better, we have expressed and characterized a heat stable, 16.6kDa Nudix hydrolase (Apf) that specifically metabolizes these nucleotides from a Drosophila melanogaster cDNA. Apf always produces an NTP product, with substrate preference depending on pH and divalent ion (Zn(2+) or Mg(2+)). For example, diadenosine tetraphosphate is hydrolysed to ATP and AMP with K(m), k(cat) and k(cat)/K(m) values 9microM, 43s(-1) and 4.8microM(-1)s(-1) (pH 6.5, 0.1mMZn(2+)) and 12microM, 13s(-1) and 1.1microM(-1)s(-1) (pH 7.5, 20mMMg(2+)), respectively. However, diadenosine hexaphosphate is efficiently hydrolysed to ATP only at pH 7.5 with 20mMMg(2+) (K(m), k(cat) and k(cat)/K(m) values of 15microM 4.0s(-1), and 0.27microM(-1)s(-1)). Fluoride potently inhibits diadenosine tetraphosphate hydrolysis in the presence of Mg(2+) (IC(50)=20microM), whereas it is ineffective in the presence of Zn(2+), supporting the view that inhibition involves a specific, MgF(3)(-)-containing transition state analogue complex. Patterns of Apf expression in Drosophila tissues show Apf mRNA levels to be highest in embryos and adult females. Subcellular localization with Apf-EGFP fusion constructs reveals Apf to be predominantly nuclear, having an apparent preferential association with euchromatin and facultative heterochromatin. This supports a nuclear function for diadenosine tetraphosphate. Our results show Apf to be a fairly typical member of the bis (5'-nucleosyl)-tetraphosphatase subfamily of Nudix hydrolases with features that distinguish it from a previously reported bis (5'-nucleosyl)-tetraphosphatase hydrolase activity from Drosophila embryos.     

The role of tyrosine 343 in substrate binding and catalysis by human sulfite oxidase

In the crystal structure of chicken sulfite oxidase, the residue Tyr(322) (Tyr(343) in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6-10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, k(cat) was somewhat impaired ( approximately 34% wild-type activity at pH 8.5), whereas the K(m)(sulfite) showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, k(red)(heme) changed very little over the entire pH range, with a significant increase in K(d)(sulfite) at high pH. The k(red)(heme) of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both k(red)(heme) and K(d)(sulfite) were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (k(red)(Mo) = 972 s(-1) at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr(343) residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase.     

Purification and characterization of a novel erythrose reductase from Candida magnoliae

Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K(m) = 7.9 mM; k(cat)/K(m) = 0.73 mM(-1) s(-1)). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k(cat)/K(m) = 450 mM(-1) s(-1)) than with NADPH (k(cat)/K(m) = 5.5 mM(-1) s(-1)), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu(2+) and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.     

Natural substrate assay for chitinases using high-performance liquid chromatography: a comparison with existing assays

The determination of kinetic parameters of chitinases using natural substrates is difficult due to low K(m) values, which require the use of low substrate concentrations that are hard to measure. Using the natural substrate (GlcNAc)(4), we have developed an assay for the determination of k(cat) and K(m)values of chitinases. Product concentrations as low as 0.5 microM were detected using normal-phase high-performance liquid chromatography (HPLC) with an amide 80 column (0.20 x 25 cm) using spectrophotometric detection at 210 nm. By means of this assay, k(cat) and K(m)values for chitinases A (ChiA) and B (ChiB) of Serratia marcescens were found to be 33+/-1s(-1) and 9+/-1 microM and 28+/-2s(-1) and 4+/-2 microM, respectively. For ChiB, these values were compared to those found with commonly used substrates where the leaving group is a (nonnatural) chromophore, revealing considerable differences. For example, assays with 4-methylumbelliferyl-(GlcNAc)(2) yielded a k(cat) value of 18+/-2s(-1) and a K(m) value of 30+/-6 microM. For two ChiB mutants containing a Trp --> Ala mutation in the +1 or +2 subsites, the natural substrate and the 4-methylumbelliferyl-(GlcNAc)(2) assays yielded rather similar K(m) values (5-fold difference at most) but showed dramatic differences in k(cat) values (up to 90-fold). These results illustrate the risk of using artificial substrates for characterization of chitinases and, thus, show that the new HPLC-based assay is a valuable tool for future chitinase research.