A covalent modification of NADP+ revealed by the atomic resolution structure of FprA,a Mycobacterium tuberculosis oxidoreductase

FprA is a mycobacterial oxidoreductase that catalyzes the transfer of reducing equivalents from NADPH to a protein acceptor. We determined the atomic resolution structure of FprA in the oxidized (1.05 A resolution) and NADPH-reduced (1.25 A resolution) forms. The comparison of these FprA structures with that of bovine adrenodoxin reductase showed no significant overall differences. Hence, these enzymes, which belong to the structural family of the disulfide oxidoreductases, are structurally conserved in very distant organisms such as mycobacteria and mammals. Despite the conservation of the overall fold, the details of the active site of FprA show some peculiar features. In the oxidized enzyme complex, the bound NADP+ exhibits a covalent modification, which has been identified as an oxygen atom linked through a carbonylic bond to the reactive C4 atom of the nicotinamide ring. Mass spectrometry has confirmed this assignment. This NADP+ derivative is likely to form by oxidation of the NADP+ adduct resulting from nucleophilic attack by an active-site water molecule. A Glu-His pair is well positioned to activate the attacking water through a mechanism analogous to that of the catalytic triad in serine proteases. The NADP+ nicotinamide ring exhibits the unusual cis conformation, which may favor derivative formation. The physiological significance of this reaction is presently unknown. However, it could assist with drug-design studies in that the modified NADP+ could serve as a lead compound for the development of specific inhibitors.     

Binding of coenzyme and substrate and coenzyme analogues to 6-phosphogluconate dehydrogenase from sheep liver. An X-ray study at 0.6 nm resolution.

The analogues of the coenzyme NADP+, nicotinamide--8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine--adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was found to be active in the hydrogen transfer adn io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP+, NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+ -NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.     

A productive NADP+ binding mode of ferredoxin-NADP + reductase revealed by protein engineering and crystallographic studies.

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom.     

The role of Cys-298 in aldose reductase function

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.     

Human glucose-6-phosphate dehydrogenase: the crystal structure reveals a structural NADP(+) molecule and provides insights into enzyme deficiency

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) catalyses the first committed step in the pentose phosphate pathway; the generation of NADPH by this enzyme is essential for protection against oxidative stress. The human enzyme is in a dimer<-->tetramer equilibrium and its stability is dependent on NADP(+) concentration. G6PD deficiency results from many different point mutations in the X-linked gene encoding G6PD and is the most common human enzymopathy. Severe deficiency causes chronic non-spherocytic haemolytic anaemia; the usual symptoms are neonatal jaundice, favism and haemolytic anaemia. RESULTS: We have determined the first crystal structure of a human G6PD (the mutant Canton, Arg459-->Leu) at 3 A resolution. The tetramer is a dimer of dimers. Despite very similar dimer topology, there are two major differences from G6PD of Leuconostoc mesenteroides: a structural NADP(+) molecule, close to the dimer interface but integral to the subunit, is visible in all subunits of the human enzyme; and an intrasubunit disulphide bond tethers the otherwise disordered N-terminal segment. The few dimer-dimer contacts making the tetramer are charge-charge interactions. CONCLUSIONS: The importance of NADP(+) for stability is explained by the structural NADP(+) site, which is not conserved in prokaryotes. The structure shows that point mutations causing severe deficiency predominate close to the structural NADP(+) and the dimer interface, primarily affecting the stability of the molecule. They also indicate that a stable dimer is essential to retain activity in vivo. As there is an absolute requirement for some G6PD activity, residues essential for coenzyme or substrate binding are rarely modified.     

Purification and subunit structure of phosphoglycerate dehydrogenase from rabbit liver.

The nicotinamide nucleotide dimers (NAD)2 and (NADP)2, obtained by electrochemical reduction of NAD+ and NADP+, are able to reduce such single-electron acceptors as the proteins cytochrome c, azurin and methaemoglobin, though at different rates. Under the same conditions the reduced nicotinamide coenzymes NADH and NADPH are not able to reduce these proteins at measurable rates unless a catalyst (phenazine methosulphate or NADH-cytochrome c reductase in the case of cytochrome) is present. The redox mechanism seems to involve the formation of an NAD(P). radical that in the presence of O2 gives rise to superoxide (O2.-), since superoxide dismutase inhibited these reactions.     

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.     

Conserved catalytic residues of the ALDH1L1 aldehyde dehydrogenase domain control binding and discharging of the coenzyme

The C-terminal domain (C(t)-FDH) of 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an NADP(+)-dependent oxidoreductase and a structural and functional homolog of aldehyde dehydrogenases. Here we report the crystal structures of several C(t)-FDH mutants in which two essential catalytic residues adjacent to the nicotinamide ring of bound NADP(+), Cys-707 and Glu-673, were replaced separately or simultaneously. The replacement of the glutamate with an alanine causes irreversible binding of the coenzyme without any noticeable conformational changes in the vicinity of the nicotinamide ring. Additional replacement of cysteine 707 with an alanine (E673A/C707A double mutant) did not affect this irreversible binding indicating that the lack of the glutamate is solely responsible for the enhanced interaction between the enzyme and the coenzyme. The substitution of the cysteine with an alanine did not affect binding of NADP(+) but resulted in the enzyme lacking the ability to differentiate between the oxidized and reduced coenzyme: unlike the wild-type C(t)-FDH/NADPH complex, in the C707A mutant the position of NADPH is identical to the position of NADP(+) with the nicotinamide ring well ordered within the catalytic center. Thus, whereas the glutamate restricts the affinity for the coenzyme, the cysteine is the sensor of the coenzyme redox state. These conclusions were confirmed by coenzyme binding experiments. Our study further suggests that the binding of the coenzyme is additionally controlled by a long-range communication between the catalytic center and the coenzyme-binding domain and points toward an α-helix involved in the adenine moiety binding as a participant of this communication.     

Oxidation of excited-state NADH and NAD dimer in aqueous medium involvement of O2− as a mediator in the presence of oxygen

It has been shown that direct excitation of NADH (or NADPH) in aqueous medium at 254 nm, or at wavelengths longer than 320 nm (where only the reduced nicotinamide moiety absorbs), leads to generation of NAD⁺ (or NADP⁺). The reaction proceeds both in the presence and absence of oxygen. Under aerobic conditions the reaction is accompanied by formation of H₂O₂ at a level equimolar with that of the NADH present in solution. On irradiation at wavelengths longer than 320 nm, conversion of NADH to enzymatically active NAD⁺ is about 75%. Under analogous irradiation conditions, the dimers (NAD)₂ and (NADP)₂ undergo disproportionation to NAD⁺ and NADP⁺, respectively, to the extent of 90%. Both physicochemical and enzymatic criteria were employed to formulate mechanisms for the photooxidation of NADH and the photodisproportionation of the dimer (NAD)₂.     

1H nuclear magnetic resonance studies of the conformation and environment of nucleotides bound to pig heart NADP+-dependent isocitrate dehydrogenase

The binding of coenzymes, NADP+ and NADPH, and coenzyme fragments, 2'-phosphoadenosine 5'-(diphosphoribose), adenosine 2',5'-bisphosphate, and 2'-AMP, to pig heart NADP+-dependent isocitrate dehydrogenase has been studied by proton NMR. Transferred nuclear Overhauser enhancement (NOE) between the nicotinamide 1'-ribose proton and the 2-nicotinamide ring proton indicates that the nicotinamide-ribose bond assumes an anti conformation. For all nucleotides, a nuclear Overhauser effect between the adenine 1'-ribose proton and 8-adenine ring proton is observed, suggesting a predominantly syn adenine--ribose bond conformation for the enzyme-bound nucleotides. Transferred NOE between the protons at A2 and N6 is observed for NADPH (but not NADP+), implying proximity between adenine and nicotinamide rings in a folded enzyme-bound form of NADPH. Line-width measurements on the resonances of free nucleotides exchanging with bound species indicate dissociation rates ranging from less than 7 s-1 for NADPH to approximately 1600 s-1 for adenosine 2',5'-bisphosphate. Substrate, magnesium isocitrate, increases the dissociation rate for NADPH about 10-fold but decreases the corresponding rate for phosphoadenosine diphosphoribose and adenosine 2',5'-bisphosphate about 10-fold. These effects are consistent with changes in equilibrium dissociation constants measured under similar conditions. The 1H NMR spectrum of isocitrate dehydrogenase at pH 7.5 has three narrow peaks between delta 7.85 and 7.69 that shift with changes in pH and hence arise from C-4 protons of histidines. One of those, with pK = 5.35, is perturbed by NADP+ and NADPH but not by nucleotide fragments, indicating that this histidine is in the region of the nicotinamide binding site. Observation of nuclear Overhauser effects arising from selective irradiation at delta 7.55 indicates proximity of either a nontitrating histidine or an aromatic residue to the adenine ring of all nucleotides. In addition, selective irradiation of the methyl region of the enzyme spectrum demonstrates that the adenine ring is close to methyl side chains. The substrate magnesium isocitrate produces no observable differences in these protein--nucleotide interactions. The alterations in enzyme--nucleotide conformation that result in changes in affinity in the presence of substrate must involve either small shifts in the positions of amino acid side chains or changes in groups not visible in the proton NMR spectrum.     

Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups.

Chemical-modification studies on submitochondrial particle pyridine dinucleotide transhydrogenase (EC 1.6.1.1) demonstrate the presence of one class of sulfhydryl group in the nicotinamide adenine dinucleotide phosphate (NADP) site and another peripheral to the active site. Reaction of the peripheral sulfhydryl group with N-ethylmaleimide, or both classes with 5,5'-dithiobis(2-nitrobenzoic acid), completely inactivated transhydrogenase. NADP+ or NADPH nearly completely protected against 5,5'-dithiobis(2-nitrobenzoic acid) inactivation and modification of both classes of sulfhydryl groups, while NADP+ only partially protected against and NADPH substantially stimulated N-ethylmaleimide inactivation. Methyl methanethiolsulfonate treatment resulted in methanethiolation at both classes of sulfhydryl groups, and either NADP+ or NADPH protected only the NADP site group. S-Methanethio and S-cyano transhydrogenases were active derivatives with pH optima shifted about 1 unit lower than that of the native enzyme. These experiments indicate that neither class of sulfhydryl group is essential for transhydrogenation. Lack of involvement of either sulfhydryl group in energy coupling to transhydrogenation is suggested by the observations that S-methanethio transhydrogenase is functional in (a) energy-linked transhydrogenation promoted by phenazine methosulfate mediated ascorbate oxidation and (b) the generation of a membrane potential during the reduction of NAD+ by reduced nicotinamide adenine dinucleotide phosphate (NADPH).     

Coenzyme binding by triphosphopyridine nucleotide dependent isocitrate dehydrogenase from beef liver. Equilibrium and kinetics studies.

The binding of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide phosphate (NADP) dependent isocitrate dehydrogenase from beef liver cytoplasm was studied by several equilibrium techniques (ultracentrifugation, molecular sieving, ultrafiltration, fluorescence). Two binding sites (per dimeric enzyme molecule) were found with slightly different dissociation constants (0.5 and 0.12 muM) and fluorescence yields (7.7 and 6.3). A ternary complex was formed between enzyme, isocitrate, and NADPH, in which NADPH dissociation constant was 5 muM. On the contrary, no binding of NADPH to the enzyme took place in the presence of magnesium isocitrate. Dialysis experiments showed the existence of 1 NADP binding site/dimer, with a dissociation constant of 26 muM. When NADPH was present with the enzyme in the proportion of 1 molecule/dimer, the dissociation constant of NADP was decreased fourfold, reaching a value quantitatively comparable to the Michaelis constant. The kinetics of coenzyme binding was followed using the stopped-flow technique with fluorescence detection. NADPH binding to the enzyme occurred through one fast reaction (k1 = 20 muM-1 s-1). Dissociation of NADPH took place upon NADP binding; however, equilibrium as well as kinetic data were incompatible with a simple competition scheme. Dissociation of NADPH from the enzyme upon magnesium isocitrate binding was preceded by the formation of a transitory ternary complex in which the fluorescence of NADPH was only about 30% of that in the enzyme-NADPH complex. Then interaction between the conenzymes and the involvement of ternary complexes in the catalytic mechanism are discussed in relation with what is known about the regulatory role of the coenzyme (Carlier, M. F., and Pantaloni, D. (1976), Biochemistry, 15, 1761-1766).     

Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase

Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2'-phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.     

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).     

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.     

Interaction of ferredoxin-NADP+ reductase from Anabaena with its substrates

The interaction of ferredoxin-NADP+ reductase from the cyanobacterium Anabaena variabilis with its substrates, NADP+ and ferredoxin, has been studied by difference absorption spectroscopy. Several structural analogs of NADP+ have been shown to form complexes the stabilities of which are strongly dependent on the ionic strength of the medium. In most cases the binding energy of these complexes and their difference absorption spectra are similar to those reported for the spinach enzyme. However, NADP+ perturbs the absorption spectra of the Anabaena and spinach enzymes in a different way. This difference has been shown to be related to the binding of the nicotinamide ring of NADP+ to the enzymes. These results are interpreted as being due to a different nicotinamide binding site in the two reductases. The enthalpic and entropic components of the Gibbs energy of formation of the NADP+ complex have been estimated. An increase in entropy on NADP+ binding seems to be the main source of stability for the complex. A shift of approximately 40 mV in the redox potential of the couple NADP+/NADPH has been observed to occur upon binding of NADP+ to the oxidized enzyme. This allows us to calculate the binding energy between the reductase and NADPH. The ability of the reductase, ferredoxin, and NADP+ to form a ternary complex indicates that the protein carrier binds to the reductase through a different site than that of the pyridine nucleotide.     

The Antarctic Psychrobacter sp. TAD1 has two cold-active glutamate dehydrogenases with different cofactor specificities. Characterisation of the NAD+-dependent enzyme

Psychrobacter sp. TAD1 is a psychrotolerant bacterium from Antarctic frozen continental water that grows from 2 to 25 degrees C with optimal growth rate at 20 degrees C. The new isolate contains two glutamate dehydrogenases (GDH), differing in their cofactor specificities, subunit sizes and arrangements, and thermal properties. NADP+-dependent GDH is a hexamer of 47 kDa subunits and it is comparable to other hexameric GDHs of family-I from bacteria and lower eukaria. The NAD+-dependent enzyme, described in this communication, has a subunit weight of 160 kDa and belongs to the novel class of GDHs with large size subunits. The enzyme is a dimer; this oligomeric arrangement has not been reported previously for GDH. Both enzymes have an apparent optimum temperature for activity of approximately 20 degrees C, but their cold activities and thermal labilities are different. The NAD+-dependent enzyme is more cold active: at 10 C it retains 50% of its maximal activity, compared with 10% for the NADP+-dependent enzyme. The NADP+-dependent enzyme is more heat stable, losing only 10% activity after heating for 30 min, compared with 95% for the NAD+-dependent enzyme. It is concluded that in Psychrobacter sp. TAD1 not only does NAD+-dependent GDH have a novel subunit molecular weight and arrangement, but that its polypeptide chains are folded differently from those of NADP+-dependent GDH, providing different cold-active properties to the two enzymes.     

Kinetic and nuclear magnetic resonance study of the interaction of NADP+ and NADPH with chicken liver fatty acid synthase

The ionic strength dependence of the second-order rate constant for the association of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and chicken liver fatty acid synthase was determined. This rate constant is 7.2 X 10(7) M-1 s-1 at zero ionic strength and 25 degrees C; the effective charge at the cofactor binding sites is +0.8. The conformations of nicotinamide adenine dinucleotide phosphate (NADP+) and NADPH bound to the beta-ketoacyl and enoyl reductase sites were determined from transferred nuclear Overhauser effect measurements. Covalent modification of the enzyme with pyridoxal 5'-phosphate abolished cofactor binding at the enoyl reductase site; this permitted the cofactor conformations at the beta-ketoacyl and enoyl reductase sites to be distinguished. For NADP+ bound to the enzyme, the conformation of the nicotinamide-ribose bond is anti at the enoyl reductase site and syn at the beta-ketoacyl reductase site; the adenine-ribose bond is anti, and the sugar puckers are C3'-endo. Nicotinamide-adenine base stacking was not detected. Structural models of NADP+ at the beta-ketoacyl and enoyl reductase sites were constructed by using the distances calculated from the observed nuclear Overhauser effects. Because of the overlap of the resonances of several nonaromatic NADPH protons with the resonances of HDO and ribose protons, less extensive structural information was obtained for NADPH bound to the enzyme. However, the conformations of NADPH bound to the two reductases are qualitatively the same as those of NADP+, except that the nicotinamide moiety of NADPH is closer to being fully anti at the enoyl reductase site.     

Kinetic models for synthesis by a thermophilic alcohol dehydrogenase

Alcohol dehydrogenase (E. C. 1.1.1.1) from Thermoanaerobium brockii at 25 degrees C and at 65 degrees C is more active with secondary than primary alcohols. The enzyme utilizes NADP and NADPH as cosubstrates better than NAD and NADH. The maximum velocities (V(m)) for secondary alcohols at 65 degrees C are 10 to 100 times higher than those at 25 degrees C, whereas the K(m) values are more comparable.At both 25 degrees C and 65 degrees C the substrate analogue 1,1,1,3,3,3-hexafluoro-2-propanol inhibited the oxidation of alcohol competitively with respect to cyclopentanol, and uncompetitively with respect to NADP. Dimethylsulfoxide inhibited the reduction of cyclopentanone competitively with respect to cyclopentanone, and uncompetitively with respect to NADPH. As a product inhibitor, NADP was competitive with respect to NADPH. These results demonstrate that the enzyme binds the nucleotide and then the alcohol or ketone to form a ternary complex which is converted to a product ternary complex that releases product and nucleotide in that order.At 25 degrees C, all aldehydes and ketones examined inhibited the enzyme at concentrations above their Michaelis constants. The substrate inhibition by cyclopentanone was incomplete, and it was uncompetitive with respect to NADPH. Furthermore, cyclopentanone as a product inhibitor showed intercept-linear, slope-parabolic inhibition with respect to cyclopentanol. These results indicate that cyclopentanone binds to the enzyme-NADP complex at high concentrations. The resulting ternary complex slowly dissociates NADP and cyclopentanone.At 65 degrees C, all of the secondary alcohols, with the exception of cyclohexanol, show substrate activation at high concentration. Experiments in which NADP was the variable substrate and cyclopentanol as the constant-variable substrate over a wide range of concentrations gave double reciprocal plots in which the intercepts showed substrate activation and the slopes showed substrate inhibition. These results indicate that the secondary alcohols bind to the enzyme-NADPH complex at high concentrations and that the resulting ternary complex dissociates NADPH faster than the enzyme-NADPH complex. (c) 1993 John Wiley & Sons, Inc.     

Acyclic monoterpene primary alcohol:NADP+ oxidoreductase of Rauwolfia serpentina cells: the key enzyme in biosynthesis of monoterpene alcohols

Acyclic monoterpene primary alcohol:NADP+ oxidoreductase, a key enzyme in the biosynthesis of monoterpene alcohols in plants, is unstable and has been only poorly characterized. However we have established conditions which stabilize the enzyme from Rauwolfia serpentina cells, and then purified it to homogeneity. It is a monomer with a molecular weight of about 44,000 and contains zinc ions. Various branched-chain allylic primary alcohols such as nerol, geraniol, and 10-hydroxygeraniol were substrates, but ethanol was inert. The enzyme exclusively requires NADP+ or NADPH as the cofactor. Steady-state kinetic studies showed that the nerol dehydrogenation proceeds by an ordered Bi-Bi mechanism. NADP+ binds the enzyme first and then NADPH is the second product released from it. Gas chromatography-mass spectrometric analysis of the reaction products showed that 10-hydroxygeraniol undergoes a reversible dehydrogenation to produce 10-oxogeraniol or 10-hydroxygeranial, which are oxidized further to give 10-oxogeranial, the direct precursor of iridodial. The enzyme has been found to exclusively transfer the pro-R hydrogen of NADPH to neral. The N-terminal sequence of the first 21 amino acids revealed no significant homology with those of various other proteins including the NAD(P)(+)-dependent alcohol dehydrogenases registered in a protein data bank.