Binding of pyridine coenzymes to the β-subunit of the voltage sensitive potassium channels

The β-subunit of the voltage-sensitive K⁺ channels shares 15–30% amino acid identity with the sequences of aldo–keto reductases (AKR) genes. However, the AKR properties of the protein remain unknown. To begin to understand its oxidoreductase properties, we examine the pyridine coenzyme binding activity of the protein in vitro. The cDNA of K_vβ2.1 from rat brain was subcloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The purified protein was tetrameric in solution as determined by size exclusion chromatography. The protein displayed high affinity binding to NADPH as determined by fluorometric titration. The K_D values for NADPH of the full-length wild-type protein and the N-terminus deleted protein were 0.1±0.007 and 0.05±0.006 M, respectively — indicating that the cofactor binding domain is restricted to the C-terminus, and is not drastically affected by the absence of the N-terminus amino acids, which form the ball and chain regulating voltage-dependent inactivation of the α-subunit. The protein displayed poor affinity for other coenzymes and the corresponding values of the K_D for NADH and NAD were between 1–3 μM whereas the K_D for FAD was >10 μM. However, relatively high affinity binding was observed with 3-acetyl pyridine NADP, indicating selective recognition of the 2′ phosphate at the binding site. The selectivity of K_vβ2.1 for NADPH over NADP may be significant in regulating the K⁺ channels as a function of the cellular redox state.     

NADPH binding to beta-subunit regulates inactivation of voltage-gated K(+) channels

Ancillary beta-subunits regulate the voltage-dependence and the kinetics of Kv currents. The Kvbeta proteins bind pyridine nucleotides with high affinity but the role of cofactor binding in regulating Kv currents remains unclear. We found that recombinant rat Kvbeta 1.3 binds NADPH (K(d)=1.8+/-0.02 microM) and NADP(+) (K(d)=5.5+/-0.9 microM). Site-specific modifications at Tyr-307 and Arg-316 decreased NADPH binding; whereas, K(d) NADPH was unaffected by the R241L mutation. COS-7 cells transfected with Kv1.5 cDNA displayed non-inactivating currents. Co-transfection with Kvbeta1.3 accelerated Kv activation and inactivation and induced a hyperpolarizing shift in voltage-dependence of activation. Kvbeta-mediated inactivation of Kv currents was prevented by the Y307F and R316E mutations but not by the R241L substitution. Additionally, the R316E mutation weakened Kvalpha-beta interaction. Inactivation of Kv currents by Kvbeta:R316E was restored when excess NADPH was included in the patch pipette. These observations suggest that NADPH binding is essential for optimal interaction between Kvalpha and beta subunits and for Kvbeta-induced inactivation of Kv currents.     

Binding of pyridine nucleotide coenzymes to the beta-subunit of the voltage-sensitive K+ channel

The beta-subunit of the voltage-sensitive K(+) (K(v)) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of K(v)beta2 shows NADP bound in its active site. Here we report that K(v)beta2 displays a high affinity for NADPH (K(d) = 0.1 micrometer) and NADP(+) (K(d) = 0.3 micrometer), as determined by fluorometric titrations of the recombinant protein. The K(v)beta2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, the K(d)(NADPH) of Q214R was 10-fold greater than the wild-type protein. The K(d)(NADPH) was unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease in K(d)(NADPH), shifted the pK(a) of K(d)(NADPH) from 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of K(v)beta2-nucleotide complexes may be formed in vivo.     

Functional interactions in cytochrome P450BM3. Evidence that NADP(H) binding controls redox potentials of the flavin cofactors

NADP(H) binding is essential for fast electron transfer through the flavoprotein domain of the fusion protein P450BM3. Here we characterize the interaction of NADP(H) with the oxidized and partially reduced enzyme and the effect of this interaction on the redox properties of flavin cofactors and electron transfer. Measurements by three different approaches demonstrated a relatively low affinity of oxidized P450BM3 for NADP(+), with a K(d) of about 10 microM. NADPH binding is also relatively weak (K(d) approximately 10 microM), but the affinity increases manyfold upon hydride ion transfer so that the active 2-electron reduced enzyme binds NADP(+) with a K(d) in the submicromolar range. NADP(H) binding induces conformational changes of the protein as demonstrated by tryptophan fluorescence quenching. Fluorescence quenching indicated preferential binding of NADPH by oxidized P450BM3, while no catalytically competent binding with reduced P450BM3 could be detected. The hydride ion transfer step, as well as the interflavin electron transfer steps, is readily reversible, as demonstrated by a hydride ion exchange (transhydrogenase) reaction between NADPH and NADP(+) or their analogues. Experiments with FMN-free mutants demonstrated that FAD is the only flavin cofactor required for the transhydrogenase activity. The equilibrium constants of each electron transfer step of the flavoprotein domain during catalytic turnover have been calculated. The values obtained differ from those calculated from equilibrium redox potentials by as much as 2 orders of magnitude. The differences result from the enzyme's interaction with NADP(H).     

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K(d) values) for NADPH (0.87 microM), NADP(+) (16 microM), NADH (50 microM), and NAD(+) (100-500 microM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K(d) values for NAD(+) and NADH are similar to those previously reported with isolated dI, but the K(d) values for NADP(+) and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidized.     

Interaction of NADP(H) with oxidized and reduced P450 reductase during catalysis. Studies with nucleotide analogues

Previous studies have shown that the interaction of P450 reductase with bound NADP(H) is essential to ensure fast electron transfer through the two flavin cofactors. In this study we investigated in detail the interaction of the house fly flavoprotein with NADP(H) and a number of nucleotide analogues. 1,4,5,6-Tetrahydro-NADP, an analogue of NADPH, was used to characterize the interaction of P450 reductase with the reduced nucleotide. This analogue is inactive as electron donor, but its binding affinity and rate constant of release are very close to those for NADPH. The 2'-phosphate contributes about 5 kcal/mol of the binding energy of NADP(H). Oxidized nicotinamide does not interact with the oxidized flavoprotein, while reduced nicotinamide contributes 1.3 kcal/mol of the binding energy. Oxidized P450 reductase binds NADPH with a K(d) of 0.3 microM, while the affinity of the reduced enzyme is considerably lower, K(d) = 1.9 microM. P450 reductase catalyzes a transhydrogenase reaction between NADPH and oxidized nucleotides, such as thionicotinamide-NADP(+), acetylpyridine-NADP(+), or [(3)H]NADP(+). The reverse reaction, reduction of [(3)H]NADP(+) by the reduced analogues, is also catalyzed by P450 reductase. We define the mechanism of the transhydrogenase reaction as follows: NADPH binding, hydride ion transfer, and release of the NADP(+) formed. An NADP(+) or its analogue binds to the two-electron-reduced flavoprotein, and the electron-transfer steps reverse to transfer hydride ion to the oxidized nucleotide, which is released. Measurements of the flavin semiquinone content, rate constant for NADPH release, and transhydrogenase turnover rates allowed us to estimate the steady-state distribution of P450 reductase species during catalysis, and to calculate equilibrium constants for the interconversion of catalytic intermediates. Our results demonstrate that equilibrium redox potentials of the flavin cofactors are not the sole factor governing rapid electron transfer during catalysis, but conformational changes must be considered to understand P450 reductase catalysis.     

Elucidation of a complete kinetic mechanism for a mammalian hydroxysteroid dehydrogenase (HSD) and identification of all enzyme forms on the reaction coordinate: the example of rat liver 3alpha-HSD (AKR1C9)

Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3alpha-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP(+) (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP(+) controlled k(cat), whereas the chemical event also contributed to this term. k(cat) was insensitive to [(2)H]NADPH, whereas (D)k(cat)/K(m) and the (D)k(lim) (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated k(cat), whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall K(eq) favored reduction. The kinetic Haldane and free energy diagram confirmed that K(eq) was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.     

Crystal structure of human liver Delta4-3-ketosteroid 5beta-reductase (AKR1D1) and implications for substrate binding and catalysis

AKR1D1 (steroid 5beta-reductase) reduces all Delta(4)-3-ketosteroids to form 5beta-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an alpha,beta-unsaturated ketone by 5beta-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the beta-face of a Delta(4)-3-ketosteroid yields a cis-A/B-ring configuration with an approximately 90 degrees bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human Delta(4)-3-ketosteroid 5beta-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP(+) at 1.79- and 1.35-A resolution (HEPES bound in the active site), NADP(+) and cortisone at 1.90-A resolution, NADP(+) and progesterone at 2.03-A resolution, and NADP(+) and testosterone at 1.62-A resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP(+). This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr(58) and Glu(120). The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.     

Lysine 219 participates in NADPH specificity in a flavin-containing monooxygenase from Saccharomyces cerevisiae

The flavin-containing monooxygenase from Saccharomyces cerevisiae (yFMO) uses NADPH and O(2) to oxidize thiol containing substrates such as GSH and thereby generates the oxidizing potential for the ER. The enzyme uses NADPH 12 times more efficiently than NADH. Amino acid sequence analysis suggests that Lys 219 and/or Lys 227 may act as counterions to the 2' phosphate of NADPH and to help determine the preference for pyridine nucleotides. Site directed mutations show that Lys 219 makes the greater contribution to cosubstrate recognition. Conversion of Lys 219 to Ala reduces NADPH dependent activity 90-fold, but has no effect on NADH-dependent activity. Conversion of Lys 227 to Ala reduces NADPH-dependent activity fivefold and NADH-dependent activity threefold. Dissociation constants for NADP(+) to oxidized yFMO were measured spectroscopically. K(d) is 12 microM for the wild-type enzyme and 243 microM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.     

Catalytic mechanism of the primary human prostaglandin F2α synthase, aldo-keto reductase 1B1--prostaglandin D2 synthase activity in the absence of NADP(H)

Aldo-keto reductase 1B1 and 1B3 (AKR1B1 and AKR1B3) are the primary human and mouse prostaglandin F(2α) (PGF(2α)) synthases, respectively, which catalyze the NADPH-dependent reduction of PGH(2), a common intermediate of various prostanoids, to form PGF(2α). In this study, we found that AKR1B1 and AKR1B3, but not AKR1B7 and AKR1C3, also catalyzed the isomerization of PGH(2) to PGD(2) in the absence of NADPH or NADP(+). Both PGD(2) and PGF(2α) synthase activities of AKR1B1 and AKR1B3 completely disappeared in the presence of NADP(+) or after heat treatment of these enzymes at 100 °C for 5 min. The K(m), V(max), pK and optimum pH values of the PGD(2) synthase activities of AKR1B1 and AKR1B3 were 23 and 18 μM, 151 and 57 nmol·min(-1)·(mg protein)(-1), 7.9 and 7.6, and pH 8.5 for both AKRs, respectively, and those of PGF(2α) synthase activity were 29 and 33 μM, 169 and 240 nmol·min(-1)·(mg protein)(-1), 6.2 and 5.4, and pH 5.5 and pH 5.0, respectively, in the presence of 0.5 mm NADPH. Site-directed mutagenesis of the catalytic tetrad of AKR1B1, composed of Tyr, Lys, His and Asp, revealed that the triad of Asp43, Lys77 and His110, but not Tyr48, acts as a proton donor in most AKR activities, and is crucial for PGD(2) and PGF(2α) synthase activities. These results, together with molecular docking simulation of PGH(2) to the crystallographic structure of AKR1B1, indicate that His110 acts as a base in concert with Asp43 and Lys77 and as an acid to generate PGD(2) and PGF(2α) in the absence of NADPH or NADP(+) and in the presence of NADPH, respectively.     

Dissection of the physiological interconversion of 5alpha-DHT and 3alpha-diol by rat 3alpha-HSD via transient kinetics shows that the chemical step is rate-determining: effect of mutating cofactor and substrate-binding pocket residues on catalysis

3Alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) catalyze the interconversion between 5alpha-dihydrotestosterone (5alpha-DHT), the most potent androgen, and 3alpha-androstanediol (3alpha-diol), a weak androgen metabolite. To identify the rate-determining step in this physiologically important reaction, rat liver 3alpha-HSD (AKR1C9) was used as the protein model for the human homologues in fluorescence stopped-flow transient kinetic and kinetic isotope effect studies. Using single and multiple turnover experiments to monitor the NADPH-dependent reduction of 5alpha-DHT, it was found that k(lim) and k(max) values were identical to k(cat), indicating that chemistry is rate-limiting overall. Kinetic isotope effect measurements, which gave (D)k(cat) = 2.4 and (D)2(O)k(cat) = 3.0 at pL 6.0, suggest that the slow chemical transformation is significantly rate-limiting. When the NADP(+)-dependent oxidation of 3alpha-diol was monitored, single and multiple turnover experiments showed a k(lim) and burst kinetics consistent with product release as being rate-limiting overall. When NAD(+) was substituted for NADP(+), burst phase kinetics was eliminated, and k(max) was identical to k(cat). Thus with the physiologically relevant substrates 5alpha-DHT plus NADPH and 3alpha-diol plus NAD(+), the slowest event is chemistry. R276 forms a salt-linkage with the phosphate of 2'-AMP, and when it is mutated, tight binding of NAD(P)H is no longer observed [Ratnam, K., et al. (1999) Biochemistry 38, 7856-7864]. The R276M mutant also eliminated the burst phase kinetics observed for the NADP(+)-dependent oxidation of 3alpha-diol. The data with the R276M mutant confirms that the release of the NADPH product is the slow event; and in its absence, chemistry becomes rate-limiting. W227 is a critical hydrophobic residue at the steroid binding site, and when it is mutated to alanine, k(cat)/K(m) for oxidation is significantly depressed. Burst phase kinetics for the NADP(+)-dependent turnover of 3alpha-diol by W227A was also abolished. In the W227A mutant, the slow release of NADPH is no longer observed since the chemical transformation is now even slower. Thus, residues in the cofactor and steroid-binding site can alter the rate-determining step in the NADP(+)-dependent oxidation of 3alpha-diol to make chemistry rate-limiting overall.     

NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity

The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR). Doubly labeled [U-(13)C,(15)N]DHFR was obtained from Escherichia coli grown on a medium containing [U-(13)C]-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin. Backbone and side chain NMR assignments were made using triple-resonance experiments. The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites. Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology. We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time. Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity. Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase. (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+). Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH. Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+). These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.     

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.     

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.     

The kinetic mechanism of the anterior pituitary progesterone 5 alpha-reductase

An analysis of the kinetic mechanism of the microsomal NADPH-linked progesterone 5 alpha-reductase obtained from female rat anterior pituitaries was performed. Initial velocity, product inhibition and dead-end inhibition studies indicate that the kinetic mechanism for the progesterone 5 alpha-reductase is equilibrium ordered sequential. Analysis of the initial velocity data resulted in intersecting double reciprocal plots suggesting a sequential mechanism [apparent Km(progesterone) = 88.2 +/- 8.2 nM; apparent Kia(NADPH) = 7.7 +/- 1.1 microM]. Furthermore, the plot of 1/v vs 1/progesterone intersected on the ordinate which is indicative of an equilibrium ordered mechanism. Additional support for ordered substrate binding was provided by the product inhibition studies with NADPH versus NADP and progesterone versus NADP. NADP is a competitive inhibitor versus NADPH (apparent Kis = 7.8 +/- 1.0 microM) and a noncompetitive inhibitor versus progesterone (apparent Kis = 9.85 +/- 2.1 microM and apparent Kii = 63.2 +/- 12.5 microM). These inhibition patterns suggest that NADPH binds prior to progesterone. In sum, these kinetic studies indicate that NADPH binds to the microsomal enzyme in rapid equilibrium and preferentially precedes the binding of progesterone.     

Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling.

PTR1, the gene promoting MTX resistance following gene amplification or DNA transfection in Leishmania tarentolae and selected mutants, has been cloned and heavily overexpressed (>100 mg/liter) in Escherichia coli strain BL21 (DE3). Protein has been purified, essentially to homogeneity, in two steps, via ammonium sulfate precipitation and chromatography on DEAE-Trisacryl. The active proteins are tetramers and display optimal pteridine reductase activity at pH 6.0 using biopterin as substrate and NADPH as the reduced dinucleotide cofactor. 2,4-Diaminopteridine substrate analogues are strong competitive inhibitors (K(i) approximately 38 --> 3 nM) against the pterin substrate and both NADP(+) and folate are inhibitors although somewhat weaker. Dihydropteridines are poor substrates compared to the fully oxidized pteridine. Kinetic analysis affords the usual Michaelis constants and in addition shows that inhibition by NADP(+) allows the formation of ternary nonproductive complexes with folate. The kinetic results are consistent with a sequential ordered bi-bi kinetic mechanism in which first NADPH and then pteridine bind to the free enzyme. Sequence comparisons suggest that PTR1 belongs to the short-chain dehydrogenase/reductase (SDR) family containing an amino-terminal glycine-rich dinucleotide binding site plus a catalytic Y(Xaa)(3)K motif. In accord with this observation, the mutants K16A, Y37D, and R39A and the double mutants K17A:R39A and Y37D:R39A all show a two- to threefold lower binding affinity for NADPH and exhibit low or zero activity. Two Y(Xaa)(3)K regions are present in wild-type PTR1 at 152 and 194. Only Y194F gives protein with zero activity. This observation coupled with affinity labeling of PTR1 by oNADP(+) (2', 3'-dialdehyde derivative of NADP(+)) followed by NaBH(4) reduction, V8 protease digestion, and mass spectral analysis suggests that the motif participating in catalysis is that at 194. The mutation K198Q eliminates inactivation by oNADP(+) supporting the hypothesis that K198 is associated with nucleotide orientation, as has been demonstrated for similar lysine residues in other members of the SDR family.     

Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily.

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.     

Evidence for the stabilization of NADPH relative to NADP(+) on the dIII components of proton-translocating transhydrogenases from Homo sapiens and from Rhodospirillum rubrum by measurement of tryptophan fluorescence.

A unique Trp residue in the recombinant dIII component of transhydrogenase from human heart mitochondria (hsdIII), and an equivalent Trp engineered into the dIII component of Rhodospirillum rubrum transhydrogenase (rrdIII.D155W), are more fluorescent when NADP(+) is bound to the proteins, than when NADPH is bound. We have used this to determine the occupancy of the binding site during transhydrogenation reactions catalysed by mixtures of recombinant dI from the R. rubrum enzyme and either hsdIII or rrdIII.D155W. The standard redox potential of NADP(+)/NADPH bound to the dIII proteins is some 60-70 mV higher than that in free solution. This results in favoured reduction of NADP(+) by NADH at the catalytic site, and supports the view that changes in affinity at the nucleotide-binding site of dIII are central to the mechanism by which transhydrogenase is coupled to proton translocation across the membrane.     

Catalytic mechanism and substrate specificity of the beta-subunit of the voltage-gated potassium channel

The beta-subunits of voltage-gated potassium (Kv) channels are members of the aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvbeta proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvbeta2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06-0.2 min(-1)) than those of most other AKRs but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvbeta2. The protein catalyzed the reduction of a broad range of carbonyls, including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipids, and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0-3.1) were observed under single- and multiple-turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvbeta proteins are catalytically active AKRs that impart redox sensitivity to Kv channels.     

Involvement of NADPH in the interaction between heme oxygenase-1 and cytochrome P450 reductase

Heme oxygenase-1 (HO-1) catalyzes the physiological degradation of heme at the expense of molecular oxygen using electrons donated by NADPH-cytochrome P450 reductase (CPR). In this study, we investigated the effect of NADP(H) on the interaction of HO-1 with CPR by surface plasmon resonance. We found that HO-1 associated with CPR more tightly in the presence of NADP(+) (K(D) = 0.5 microm) than in its absence (K(D) = 2.4 microm). The HO-1 mutants, K149A, K149A/K153A, and R185A, showed almost no heme degradation activity with NADPH-CPR, whereas they exhibited activity comparable to that of the wild type when sodium ascorbate was used. R185A showed a 100-fold decreased affinity for CPR compared with wild type, even in the presence of NADP(+) (K(D) = 36.3 microm). The affinities of K149A and K149A/K153A for CPR were decreased 7- and 9-fold (K(D) = 16.8 and 21.8 microm), respectively. In contrast to R185A, the affinities of K149A and K149A/K153A were improved by the addition of NADP(+) (K(D) = 5.2 and 9.6 microm, respectively), as was the case with wild type. Computer modeling of the HO-1/CPR complex showed that the guanidino group of Arg(185) is located within the hydrogen bonding distance of 2'-phosphate of NADPH, suggesting that Arg(185) contributes to the binding to CPR through an electrostatic interaction with the phosphate group. On the other hand, Lys(149) is close to a cluster of acidic amino acids near the FMN binding site of CPR. Thus, Lys(149) and Lys(153) appear to interact with CPR in such a way as to orient the redox partners for optimal electron transfer from FMN of CPR to heme of HO-1.