Phosphate Of NADPH Research Articles

NADH/NADPH bi-cofactor-utilizing and thermoactive ketol-acid reductoisomerase from Sulfolobus acidocaldarius

Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of branched-chain amino acids biosynthetic pathway. Most KARIs prefer NADPH as a cofactor. However, KARI with a preference for NADH is desirable in industrial applications including anaerobic fermentation for the production of branched-chain amino acids or biofuels. Here, we characterize a thermoacidophilic archaeal Sac-KARI from Sulfolobus acidocaldarius and present its crystal structure at a 1.75-Å resolution. By comparison with other holo-KARI structures, one sulphate ion is observed in each binding site for the 2′-phosphate of NADPH, implicating its NADPH preference. Sac-KARI has very high affinity for NADPH and NADH, with KM values of 0.4 μM for NADPH and 6.0 μM for NADH, suggesting that both are good cofactors at low concentrations although NADPH is favoured over NADH. Furthermore, Sac-KARI can catalyze 2(S)-acetolactate (2S-AL) with either cofactor from 25 to 60 °C, but the enzyme has higher activity by using NADPH. In addition, the catalytic activity of Sac-KARI increases significantly with elevated temperatures and reaches an optimum at 60 °C. Bi-cofactor utilization and the thermoactivity of Sac-KARI make it a potential candidate for use in metabolic engineering or industrial applications under anaerobic or harsh conditions.

Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA

Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2′-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP+ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation.

Development of a Bioconversion System Using Saccharomyces cerevisiae Reductase YOR120W and Bacillus subtilis Glucose Dehydrogenase for Chiral Alcohol Synthesis

Reductases convert some achiral ketone compounds into chiral alcohols, which are important materials for the synthesis of chiral drugs. The Saccharomyces cerevisiae reductase YOR120W converts ethyl-4-chloro-3-oxobutanoate (ECOB) enantioselectively into (R)-ethyl-4-chloro-3- hydroxybutanoate ((R)-ECHB), an intermediate of a pharmaceutical. As YOR120W requires NADPH as a cofactor for the reduction reaction, a cofactor recycling system using Bacillus subtilis glucose dehydrogenase was employed. Using this coupling reaction system, 100 mM ECOB was converted to (R)-ECHB. A homology modeling and site-directed mutagenesis experiment were performed to determine the NADPH-binding site of YOR120W. Four residues (Q29, K264, N267, and R270) were suggested by homology and docking modeling to interact directly with 2'-phosphate of NADPH. Among them, two positively charged residues (K264 and R270) were experimentally demonstrated to be necessary for NADPH 2'-phosphate binding. A mutant enzyme (Q29E) showed an enhanced enantiomeric excess value compared with that of the wild-type enzyme.

Computational design of short-chain dehydrogenase Gox2181 for altered coenzyme specificity

Short-chain dehydrogenase Gox2181 from Gluconobacter oxydans catalyzes the reduction of 2,3-pentanedione by using NADH as the physiological electron donor. To realize its synthetic biological application for coenzyme recycling use, computational design and site-directed mutagenesis have been used to engineer Gox2181 to utilize not only NADH but also NADPH as the electron donor. Single and double mutations at residues Q20 and D43 were made in a recombinant expression system that corresponded to Gox2181-D43Q and Gox2181-Q20R&D43Q, respectively. The design of mutant Q20R not only resolved the hydrogen bond interaction and electrostatic interaction between R and 2′-phosphate of NADPH, but also could enhance the binding with 2′-phophated of NADPH by combining with D43Q. Molecular dynamics simulation has been carried out to testify the hydrogen bond interactions between mutation sites and 2′-phosphate of NADPH. Steady-state turnover measurement results indicated that Gox2181-D43Q could use both NADH and NADPH as its coenzyme, and so could Gox2181-Q20R&D43Q. Meanwhile, compared to the wild-type enzyme, Gox2181-D43Q exhibited dramatically reduced enzymatic activity while Gox2181-Q20R&D43Q successfully retained the majority of enzymatic activity.

The effect of pH on the initial rate kinetics of the dimeric biliverdin‐IXα reductase from the cyanobacterium Synechocystis PCC6803

Biliverdin-IXalpha reductase from Synechocystis PCC6803 (sBVR-A) is a stable dimer and this behaviour is observed under a range of conditions. This is in contrast to all other forms of BVR-A, which have been reported to behave as monomers, and places sBVR-A in the dihydrodiol dehydrogenase/N-terminally truncated glucose-fructose oxidoreductase structural family of dimers. The cyanobacterial enzyme obeys an ordered steady-state kinetic mechanism at pH 5, with NADPH being the first to bind and NADP(+) the last to dissociate. An analysis of the effect of pH on k(cat) with NADPH as cofactor reveals a pK of 5.4 that must be protonated for effective catalysis. Analysis of the effect of pH on k(cat)/K(m)(NADPH) identifies pK values of 5.1 and 6.1 in the free enzyme. Similar pK values are identified for biliverdin binding to the enzyme-NADPH complex. The lower pK values in the free enzyme (pK 5.1) and enzyme-NADPH complex (pK 4.9) are not evident when NADH is the cofactor, suggesting that this ionizable group may interact with the 2'-phosphate of NADPH. His84 is implicated as a crucial residue for sBVR-A activity because the H84A mutant has less than 1% of the activity of the wild-type and exhibits small but significant changes in the protein CD spectrum. Binding of biliverdin to sBVR-A is conveniently monitored by following the induced CD spectrum for biliverdin. Binding of biliverdin to wild-type sBVR-A induces a P-type spectrum. The H84A mutant shows evidence for weak binding of biliverdin and appears to bind a variant of the P-configuration. Intriguingly, the Y102A mutant, which is catalytically active, binds biliverdin in the M-configuration.

Structure of 2,6-Dihydroxypyridine 3-hydroxylase from a Nicotine-degrading Pathway

The enzyme 2,6-dihydroxypyridine-3-hydroxylase catalyzes the sixth step of the nicotine degradation pathway in Arthrobacter nicotinovorans. The enzyme was produced in Escherichia coli, purified and crystallized. The crystal structure was solved at 2.6 Å resolution, revealing a significant structural relationship with the family of FAD-dependent aromatic hydroxylases, but essentially no sequence homology. The structure was aligned with those of the established family members, showing that the FAD molecules are bound at virtually identical locations. The reported enzyme is a dimer like most other family members, but its dimerization contact differs from the others. The binding position of NAD(P)H to this enzyme family is not clear. Since the reported enzyme accepts only NADH for flavin reduction in contrast to the other established members using NADPH, we searched through the structural alignment and found an indication for the position of the 2′-phosphate of NADPH that is in general agreement with mutational studies on a related enzyme, but contradicts a crystal soaking experiment. Using a bound glycerol molecule and the known substrate positions of three related enzymes as a guide, the substrate 2,6-dihydroxypyridine was placed into the active center. The access to the binding site is discussed. The new active center geometry introduces constraints that render some reaction scenarios more likely than others. It suggests that flavin is reduced at its out-position and then drawn into its in-position, where it binds molecular oxygen. The geometry is consistent with the proposal that peroxy-flavin is protonated by the solvent to yield the electrophilic hydroperoxy-flavin. The substrate is activated by two buried histidines but there is no appropriate base to store the surplus proton of the hydroxylated carbon atom. The implications of this problem are discussed.

Activation of biliverdin-IXα reductase by inorganic phosphate and related anions

The effect of pH on the initial-rate kinetic behaviour of BVR-A (biliverdin-IXalpha reductase) exhibits an alkaline optimum with NADPH as cofactor, but a neutral optimum with NADH as cofactor. This has been described as dual cofactor and dual pH dependent behaviour; however, no mechanism has been described to explain this phenomenon. We present evidence that the apparent peak of activity observed at neutral pH with phosphate buffer and NADH as cofactor is an anion-dependent activation, where inorganic phosphate apparently mimics the role played by the 2'-phosphate of NADPH in stabilizing the interaction between NADH and the enzyme. The enzymes from mouse, rat and human all exhibit this behaviour. This behaviour is not seen with BVR-A from Xenopus tropicalis or the ancient cyanobacterial enzyme from Synechocystis PCC 6803, which, in addition to being refractory to activation by inorganic phosphate, are also differentiated by an acid pH optimum with both nicotinamide nucleotides.

Coenzyme Binding during Catalysis Is Beneficial for the Stability of 4-Hydroxyacetophenone Monooxygenase

The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP(+) only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP(+) interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP(+)) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes.

Involvement of NADP(H) 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.

Identifying determinants of NADPH specificity in Baeyer–Villiger monooxygenases

The Baeyer-Villiger monooxygenase (BVMO), 4-hydroxyacetophenone monooxygenase (HAPMO), uses NADPH and O(2) to oxidize a variety of aromatic ketones and sulfides. The FAD-containing enzyme has a 700-fold preference for NADPH over NADH. Sequence alignment with other BVMOs, which are all known to be selective for NADPH, revealed three conserved basic residues, which could account for the observed coenzyme specificity. The corresponding residues in HAPMO (Arg339, Lys439 and Arg440) were mutated and the properties of the purified mutant enzymes were studied. For Arg440 no involvement in coenzyme recognition could be shown as mutant R440A was totally inactive. Although this mutant could still be fully reduced by NADPH, no oxygenation occurred, indicating that this residue is crucial for completing the catalytic cycle of HAPMO. Characterization of several Arg339 and Lys439 mutants revealed that these residues are indeed both involved in coenzyme recognition. Mutant R339A showed a largely decreased affinity for NADPH, as judged from kinetic analysis and binding experiments. Replacing Arg339 also resulted in a decreased catalytic efficiency with NADH. Mutant K439A displayed a 100-fold decrease in catalytic efficiency with NADPH, mainly caused by an increased K(m). However, the efficiency with NADH increased fourfold. Saturation mutagenesis at position 439 showed that the presence of an asparagine or a phenylalanine improves the catalytic efficiency with NADH by a factor of 6 to 7. All Lys439 mutants displayed a lower affinity for AADP(+), confirming a role of the lysine in recognizing the 2'-phosphate of NADPH. The results obtained could be extrapolated to the sequence-related cyclohexanone monooxygenase. Replacing Lys326 in this BVMO, which is analogous to Lys439 in HAPMO, again changed the coenzyme specificity towards NADH. These results indicate that the strict NADPH dependency of this class of monooxygenases is based upon recognition of the coenzyme by several basic residues.

The three-dimensional structures of the Mycobacterium tuberculosis dihydrodipicolinate reductase-NADH-2,6-PDC and -NADPH-2,6-PDC complexes. Structural and mutagenic analysis of relaxed nucleotide specificity.

Dihydrodipicolinate reductase (DHPR) catalyzes the reduced pyridine nucleotide-dependent reduction of the alpha,beta-unsaturated cyclic imine, dihydrodipicolinate, to generate tetrahydrodipicolinate. This enzyme catalyzes the second step in the bacterial biosynthetic pathway that generates meso-diaminopimelate, a component of bacterial cell walls, and the amino acid L-lysine. The Mycobacterium tuberculosis dapB-encoded DHPR has been cloned, expressed, purified, and crystallized in two ternary complexes with NADH or NADPH and the inhibitor 2,6-pyridinedicarboxylate (2,6-PDC). The structures have been solved using molecular replacement strategies, and the DHPR-NADH-2,6-PDC and DHPR-NADPH-2,6-PDC complexes have been refined against data to 2.3 and 2.5 A, respectively. The M. tuberculosis DHPR is a tetramer of identical subunits, with each subunit composed of two domains connected by two flexible hinge regions. The N-terminal domain binds pyridine nucleotide, while the C-terminal domain is involved in both tetramer formation and substrate/inhibitor binding. The M. tuberculosis DHPR uses NADH and NADPH with nearly equal efficiency based on V/K values. To probe the nature of this substrate specificity, we have generated two mutants, K9A and K11A, residues that are close to the 2'-phosphate of NADPH. These two mutants exhibit decreased specificity for NADPH by factors of 6- and 30-fold, respectively, but the K11A mutant exhibits 270% of WT activity using NADH. The highly conserved structure of the nucleotide fold may permit other enzyme's nucleotide specificity to be altered using similar mutagenic strategies.

Abstracts

Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2′-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP+ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation.

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 O2 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. Kd is 12 μM for the wild-type enzyme and 243 μM for the K219A mutant, consistent with the role of Lys 219 in pyridine nucleotide binding.

Probing the kinetic mechanism and coenzyme specificity of glutathione reductase from the cyanobacterium Anabaena PCC 7120 by redesign of the pyridine-nucleotide-binding site.

Glutathione reductase from the cyanobacterium Anabaena PCC 7120 contains a pyridine-nucleotide-binding motif differing from that of the enzyme from other sources and an insertion of 10 amino acid residues. Homology modeling was used to obtain a model of the enzyme structure. It revealed that in the Anabaena enzyme Lys(203) replaces Arg, found to interact with the 2'-phosphate of NADP(H) in the enzyme from other sources, and that it has an extra loop near the entrance of the pyridine-nucleotide-binding site. The steady-state and preequilibrium kinetic properties were characterized for the wild-type enzyme, a K203R, and a loop deletion mutant. All enzyme forms had higher catalytic efficiency with NADPH than with NADH, although the difference was less than for glutathione reductase from other sources. The specificity was most pronounced in the formation of the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD, as compared to later steps in the reaction. Unexpectedly, by replacing Lys(203) with Arg, the specificity for NADPH was diminished in the complete redox reaction. Ser(174) appears to interact with the 2'-phosphate of NADPH and introduction of arginine instead of lysine, therefore, has little effect on the interaction with this coenzyme. However, the efficiency in forming the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD was increased in the K203R mutant using NADPH but not with NADH. The lack of affinity toward 2',5'-ADP-Sepharose by the wild-type enzyme was not changed by replacing Lys(203) with Arg but deletion of the loop resulted in an enzyme that bound to the immobilized ligand. Removal of the loop increased the efficiency of the enzyme in the reductive half-reaction with both pyridine-nucleotides as well as in the overall catalytic mechanism.

The Arginine 276 Anchor for NADP(H) Dictates Fluorescence Kinetic Transients in 3α-Hydroxysteroid Dehydrogenase, a Representative Aldo−Keto Reductase

Fluorescence stopped-flow studies were conducted with recombinant rat liver 3 alpha-HSD, an aldo-keto reductase (AKR) that plays critical roles in steroid hormone inactivation, to characterize the binding of nicotinamide cofactor, the first step in the kinetic mechanism. Binding of NADP(H) involved two events: the fast formation of a loose complex (E.NADP(H)), followed by a conformational change in enzyme structure leading to a tightly bound complex (E.NADP(H)), which was observed as a fluorescence kinetic transient. Binding of NAD(H) was not characterized by a similar kinetic transient, implying a difference in the mode of binding of the two cofactors. Unlike previously characterized AKRs, the rates associated with the formation and decay of E.NADP(H) and E.NADP(H) were much faster than kcat for the oxidoreduction of various substrates, indicating that binding and release of cofactor is not rate-limiting overall in 3 alpha-HSD. Mutation of Arg 276, a highly conserved residue in AKRs that forms a salt bridge with the adenosine 2'-phosphate of NADP(H), resulted in large changes in Km and Kd for NADP(H) that were not observed with NAD(H). The loss in free energy associated with the increase in Kd for NADP(H) is consistent with the elimination of an electrostatic link. Importantly, this mutation abolished the kinetic transient associated with NADPH binding. Thus, anchoring of the adenosine 2'-phosphate of NADPH by Arg 276 appears to be obligatory for the fluorescence kinetic transients to be observed. The removal of Trp 86, a residue involved in fluorescence energy transfer with NAD(P)H, also abolished the kinetic transient, but mutation of Trp 227, a residue on a mobile loop associated with cofactor binding, did not. It is concluded that in 3 alpha-HSD, the time dependence of the change in Trp 86 fluorescence is due to cofactor anchoring, and thus, Trp 86 is a distal reporter of this event. Further, the loop movement that accompanies cofactor binding is spectrally silent.

Engineering of Pyridine Nucleotide Specificity of Nitrate Reductase: Mutagenesis of Recombinant CytochromebReductase Fragment ofNeurospora crassaNADPH:Nitrate Reductase

The cytochromebreductase fragment ofNeurospora crassaNADPH:nitrate reductase (EC 1.6.6.3) was overexpressed inEscherichia coliwith a His-tag for purification after mutation of the NADPH binding site. The recombinant enzyme fragment was altered by site-directed mutagenesis guided by the three-dimensional structure of cytochromebreductase fragment of corn NADH:nitrate reductase (EC 1.6.6.1). Substitution of Asp for Ser920 (using residue numbering for holo-NADPH:nitrate reductase ofN. crassa) greatly increased preference for NADH. This mutant had nearly the same NADH:ferricyanide reductasekcatas wild-type with NADPH. Substitutions for Arg921 had little influence on coenzyme specificity, while substitution of Ser or Gln for Arg932 did. The cytochromebreductase mutant with greatest preference for NADH over NADPH was the doubly substituted form, Asp for Ser920/Ser for Arg932, but it had low activity and low affinity for coenzymes, which indicated a general loss of specificity in the binding site. Steady-state kinetic constants were determined for wild type and mutants with NADPH and NADH. Wild type had a specificity ratio of 1100, which was defined as the catalytic efficiency (kcat/Km) for NADPH divided by catalytic efficiency for NADH, while Asp for Ser920 mutant had a ratio of 0.17. Thus, the specificity ratio was reversed by over 6000-fold by a single mutation. Preference for NADPH versus NADH is strongly influenced by presence/absence of a negatively charged amino acid side chain in the binding site for the 2′ phosphate of NADPH in nitrate reductase, which may partially account for existence of bispecific NAD(P)H:nitrate reductases (EC 1.6.6.2).

Switch of Coenzyme Specificity of Mouse Lung Carbonyl Reductase by Substitution of Threonine 38 with Aspartic Acid

Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, exhibits coenzyme specificity for NADP(H) over NAD(H). Crystal structure of the enzyme-NADPH complex shows that Thr-38 interacts with the 2'-phosphate of NADPH and occupies the position spatially similar to an Asp residue of the NAD(H)-dependent SDRs that hydrogen-bonds to the hydroxyl groups of the adenine ribose of the coenzymes. Using site-directed mutagenesis, we constructed a mutant mouse lung carbonyl reductase in which Thr-38 was replaced by Asp (T38D), and we compared kinetic properties of the mutant and wild-type enzymes in both forward and reverse reactions. The mutation resulted in increases of more than 200-fold in the Km values for NADP(H) and decreases of more than 7-fold in those for NAD(H), but few changes in the Km values for substrates or in the kcat values of the reactions. NAD(H) provided maximal protection against thermal and urea denaturation of T38D, in contrast to the effective protection by NADP(H) for the wild-type enzyme. Thus, the single mutation converted the coenzyme specificity from NADP(H) to NAD(H). Calculation of free energy changes showed that the 2'-phosphate of NADP(H) contributes to its interaction with the wild-type enzyme. Changing Thr-38 to Asp destabilized the binding energies of NADP(H) by 3.9-4.5 kcal/mol and stabilized those of NAD(H) by 1.2-1.4 kcal/mol. These results indicate a significant role of Thr-38 in NADP(H) binding for the mouse lung enzyme and provide further evidence for the key role of Asp at this position in NAD(H) specificity of the SDR family proteins.

Involvement of two basic residues (Lys-17 and Arg-39) of mouse lung carbonyl reductase in NADP(H)-binding and fatty acid activation: site-directed mutagenesis and kinetic analyses.

Mouse lung carbonyl reductase, a member of the short-chain dehydrogenase/reductase (SDR) family, shows a strong coenzyme preference for NADP(H) over NAD(H), and is uniquely activated by fatty acids. Previous chemical modification and X-ray crystallography studies show that interactions responsible for the coenzyme specificity include salt linkages between the 2'-phosphate of NADPH and side-chains of Lys-17 and Arg-39 of the enzyme. Although Arg-39 is highly conserved in NADP(H)-dependent enzymes of the SDR family, Lys-17 is substituted with Arg in about half of the NADP(H)-dependent enzymes. The present study shows that mutations of Lys-17 to His (K17H) or Ser (K17S) and of Arg-39 to Ala (R39A) bring about decreases (from 5 to 90-fold) of the affinities for NADP(H), but minor changes in the affinity for NAD+. The binding energy arising from the mutations on the binding of the 2'-phosphate of NADP+ was decreased by 38-66% from the value of 4.8 kcal/mol calculated for the wild-type enzyme. In contrast, the mutation of Lys-17 to Arg (K17R) had little effect on the kinetic or thermodynamic properties. The activation by fatty acids was completely attenuated by the mutations of K17H and K17S, but not by K17R or R39A. These results indicate that the 2'-phosphate group of NADP(H) is recognized by both Lys-17 and Arg-39, of which Lys-17 is a component of the binding site for the activator, probably interacting with the negatively charged carboxylate group of fatty acids, and also suggest that the existence of a positively charged residue (either Lys or Arg) at position 17 is required for both NADP(H) specificity of the SDR family enzymes and fatty acid activation of the pulmonary carbonyl reductase.

Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a p K of 8.9–9.6. The V K for both nucleotides decreased below a p K of 5.7–6.2, as did the p K i of competitive inhibitors NADP and ATP-ribose, suggesting that the 2′-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The p K of the 2′-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2–5.4. The V K for NADPH, the V K for 3-APADPH, and the p K i of ATP-ribose also decreased above a p K of 9–10, suggesting interaction of the 2′-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a p K of 8 (evident in V K for dl-glyceraldehyde and V K for l-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a p K of 9.94 facilitates the binding of carboxylic substrates such as d-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V K for 3-APADPH, and V K for d-glucuronate were pH independent and equal to 2.2–2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.

Role of lysine-54 in determining cofactor specificity and binding in human dihydrofolate reductase

Lysine-54 of human dihydrofolate reductase (hDHFR) appears to be involved in the interaction with the 2'-phosphate of NADPH and is conserved as a basic residue in other species. Studies have suggested that in Lactobacillus casei dihydrofolate reductase Arg-43, the homologous residue at this position, plays an important role in the binding of NADPH and in the differentiation of Km values for NADPH and NADH. A Lys-54 to Gln-54 mutant (K54Q) of hDHFR has been constructed by oligodeoxynucleotide-directed mutagenesis in order to study the role of Lys-54 in differentiating Km and Kcat values for NADPH and NADH as well as in other functions of hDHFR. The purpose of this paper is to delineate in quantitative terms the magnitude of the effect of the Lys-54 to Gln-54 replacement on the various kinetic parameters of hDHFR. Such quantitative effects cannot be predicted solely on the basis of X-ray structures. The Km for NADPH for the K54Q mutant enzyme is 58-fold higher, while the Km for NADH for K54Q is only 3.9-fold higher than that of the wild type, indicating that the substitution of Lys-54 with Gln-54 decreases the apparent affinity of the enzyme for NADPH dramatically, but has a lesser effect on the apparent affinity for NADH.(ABSTRACT TRUNCATED AT 250 WORDS)