Unique coenzyme binding mode of hyperthermophilic archaeal sn‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis

A gene encoding an sn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V_max) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2’ of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild‐type enzyme with NADPH, which suggests that the biased interactions around the C2’‐phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)‐dependent dehydrogenases. Proteins 2016; 84:1786–1796. © 2016 Wiley Periodicals, Inc.     

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.     

Effects of hydrogen peroxide upon nicotinamide nucleotide metabolism in Escherichia coli: changes in enzyme levels and nicotinamide nucleotide pools and studies of the oxidation of NAD(P)H by Fe(III)

DNA is damaged in vivo by the Fenton reaction mediated by Fe2+ and cellular reductants such as NADH, which reduce Fe3+ to Fe2+ and allow the recycling of iron. To study the response of Escherichia coli to such cycling, the activities of several enzymes involved in nicotinamide nucleotide metabolism were measured following an H2O2 challenge. NADPH-dependent peroxidase, NADH/NADP+ transhydrogenase, and glucose-6-phosphate dehydrogenase were most strongly induced, increasing 2.5-3-fold. In addition, the cellular ratios of NADPH to NADH increased 6- or 92-fold 15 min after exposure to 0.5 or 5 mm H2O2, respectively. In vitro, NADH was oxidized by Fe3+ up to 16-fold faster than NADPH, despite their identical reduction potentials. To understand this rate difference, the interactions of Fe3+ and Ga3+ with NAD(P)H were examined by 1H, 13C, and 31P NMR spectroscopy. Association with NADH occurred primarily with adenine at N7 and the amino group, but for NADPH, strong metal interactions also occurred at the 2'-phosphate group. Interaction of M3+ (Fe3+ or Ga3+) with the adenine ring would bring it into close proximity to the redox-active nicotinamide ring in the folded form of NAD(P)H, but interaction of M3+ with the 2'-phosphate group would avoid this close contact. In addition, as determined by absorbance spectroscopy, the energy of the charge-transfer species was significantly higher for the Fe3+.NADPH complex than for the Fe3+.NADH complex. We therefore suggest that upon exposure to H2O2 the NADH pool is depleted, and NADPH, which is less reactive with Fe3+, functions as the major nicotinamide nucleotide reductant.     

Combination of NADPH and copper ions generates proteinase K-resistant aggregates from recombinant prion protein

Recent studies have demonstrated that the octapeptide repeats of the N-terminal region of prion protein may be responsible for de novo generation of infectious prions in the absence of template. Here we demonstrate that PrP-(23-98), an N-terminal portion of PrP, is converted to aggregates upon incubation with NADPH and copper ions. Other pyridine nucleotides possessing a phosphate group on the adenine-linked ribose moiety (the reduced form of nicotinamide adenine dinucleotide 3'-phosphate, nicotinic acid adenine dinucleotide phosphate, and NADP) were also effective in promoting aggregation, but NADH and NAD had no effect. The aggregation was attenuated by the metal chelator EDTA or by modification of histidyl residues with diethyl pyrocarbonate. The aggregates are amyloid-like as judged by the binding of thioflavin T, a fluorescent probe for amyloid, but do not exhibit fibrillar structures according to electron micrography. Interestingly the aggregates were resistant to proteinase K digestion. Likewise NADPH and zinc ions caused aggregation of PrP-(23-98), but the resulting aggregates were susceptible to degradation by proteinase K. Upon incubation with NADPH and copper ions, the full-length molecule PrP-(23-231) also formed proteinase K-resistant amyloid-like aggregates. Because it is possible that PrP, NADPH, and copper ions could associate in certain tissues, the aggregation observed in this study may be involved in prion initiation especially in the nonfamilial types of prion diseases.     

Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement.

Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined. Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH. Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant. The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant. No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme. However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type. NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes. Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases. This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex.     

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.     

NADP+ expels both the co-factor and a substrate analog from the Mycobacterium tuberculosis ThyX active site: opportunities for anti-bacterial drug design

The novel flavin-dependent thymidylate synthase, ThyX, is absent in humans but several pathogenic bacteria depend exclusively on ThyX activity to synthesize thymidylate. Reduction of the enzyme-bound FAD by NADPH is suggested to be the critical first step in ThyX catalysis. We soaked Mycobacterium tuberculosis ThyX-FAD-BrdUMP ternary complex crystals in a solution containing NADP+ to gain structural insights into the reductive step of the catalytic cycle. Surprisingly, the NADP+ displaced both FAD and BrdUMP from the active site. In the resultant ThyX-NADP+ binary complex, the AMP moiety is bound in a deep pocket similar to that of the same moiety of FAD in the ternary complex, while the nicotinamide part of NADP+ is engaged in a limited number of contacts with ThyX. The additional 2'-phosphate group attached to the AMP ribose of NADP+ could be accommodated with minor rearrangement of water molecules. The newly introduced 2'-phosphate groups are engaged in water-mediated interactions across the non-crystallographic 2-fold axis of the ThyX tetramer, suggesting possibilities for design of high-affinity bivalent inhibitors of this intriguing enzyme.     

Refined X-ray structure of the low pH form of ribonuclease T1-2'-guanylic acid complex at 1.9 A resolution

The three-dimensional X-ray structure of the RNase T1[EC 3.1.27.3]-2'GMP complex crystallized at low pH value (4.0) was determined, and refined to 1.9 A resolution to give a final R value of 0.203. The refined model includes 781 protein atoms, 24 inhibitor atoms, and 43 solvent molecules. The imidazole rings of His27 and His40 interact with the carboxyl side chains of Glu82 and Glu58, respectively, whereas that of His92 is in contact with the main chain carbonyl oxygen of Ala75. In the complex, the ribose ring of the 2'GMP molecule adopts a C2'-endo puckering, and the exocyclic conformation is gauche(-)-gauche(+). The glycosyl torsion angle is in the syn range with an intramolecular hydrogen bond between N3 and O5', and the 2'-phosphate orientation is trans-gauche(-). The guanine base of the inhibitor is tightly bound to the base recognition site with five hydrogen bonds (N1--Glu46O epsilon 2, N2---Asn98O,O6---Asn44N, and N7 ---Asn43N delta 2/Asn43N) and is sandwiched between the phenolic ring portions of Tyr42 and Tyr45 by stacking interactions. The 2'-phosphate group interacts with Arg77N eta 2, Glu58O episilon 2, and Tyr 38O eta but not with any of the histidine residues. Arg77N eta 2 also interacts with Tyr38O eta. There is no interaction between the ribose moiety of the inhibitor and the enzyme.     

Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide. We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain. Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture. Introduction of a basic residue at position 34 increased the NADPH binding strength. Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity. By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2'-hydroxy group of NADH. This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase.     

Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP⁺, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.     

Probing the NADH- and Methyl Red-binding site of a FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

AzoA from Enterococcus faecalis is a member of the polymeric flavin-dependent NADH-preferred azoreductase group. Little is known about the binding and interaction of NADH and azo dye in the azoreductase group. A synergetic strategy based on computational prediction, reverse genetics validation coupled with site-directed mutagenesis, and reconstruction of mutation network was used to investigate the binding and interaction of NADH and a model azo dye, Methyl Red, with AzoA. Methyl Red and NADH interacted in a unique binding mode in which the benzoic acid moiety of Methyl Red and the nicotinamide ring of NADH were not parallel to the flavin isoalloxazine ring, but lay against it at angles of ～45° and ～35°, respectively. The adenine ribose moiety of NADH was surrounded by loop ?2 on chain B and α3 on chain A in a typical Rossmann fold. There were 12 and 19 amino acid residues that could participate in the binding of Methyl Red and NADH, respectively, especially the residues Tyr-129 and Asp-184. The functional perturbation effects of 13 residues, including Tyr-129 and Asp-184, were mapped to reconstruct the mutation network, which confirmed the proposed binding modes and also provided insights into the interaction among NADH, FMN and Methyl Red.     

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.     

Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.     

The B' helix determines cytochrome P450nor specificity for the electron donors NADH and NADPH

Nitric oxide reductase (Nor) cytochrome P450nor (P450nor) is unique because it is catalytically self-sufficient, receiving electrons directly from NADH or NADPH. However, little is known about the direct binding of NADH to cytochrome. Here, we report that oxidized pyridine nucleotides (NAD(+) and NADP(+)) and an analogue induce a spectral perturbation in bound heme when mixed with P450nor. The P450nor isoforms are classified according to electron donor specificity for NADH or NADPH. One type (Fnor, a P450nor of Fusarium oxysporum) utilizes only NADH. We found that NAD(+) induced a type I spectral change in Fnor, whereas NADP(+) induced a reverse type I spectral change, although the K(d) values for both were comparable. In contrast, NADP(+) as well as NAD(+) caused a type I spectral change in Tnor, a P450nor isozyme from Trichosporon cutaneum that utilizes both NADH and NADPH as electron donors. The B' helix region of Tnor ((73)SAGGKAAA(80)) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor ((73)SASGKQAA(80)). A single mutation (S75G) significantly improved the NADPH- dependent Nor activity of Fnor, and the overall activity was accelerated via the NADPH-enhanced reduction step. These results showed that pyridine nucleotide cofactors can bind P450nor and that only a few residues in the B' helix region determine cofactor specificity. We further showed that a poor electron donor (NADPH) could also bind Fnor, but an appropriate configuration for electron transfer is blocked by steric hindrance mainly by Ser(75) against the 2'-phosphate moiety. The present results along with previous observations together revealed a novel motif for cofactor binding.     

Computer modeling studies on the subsite interactions of ribonuclease T1.

The modes of binding of pGp,ApG,CpG and UpG to the enzyme ribonuclease T1 were determined by computer modeling. Essentially two binding modes are possible for all the four ligands--one with the 3'-phosphate group occupying the phosphate binding site (substrate mode of binding) and the second with the 5'-phosphate group occupying the phosphate binding site (inhibitor mode of binding). The latter binding mode is energetically favoured over the former and in this mode the base (G) and the 5'-phosphate moieties occupy the same sites on the enzyme as 5'-GMP when bound to RNase T1. The ribose moiety of pGp adopts a C3'-endo pucker form when bound to the enzyme and the glycosyl torsion angle will be in -syn range as 5'-GMP in the RNase T1-5'-GMP complex. Based on these results, a mechanism for the release of the product subsequent to cleavage of the substrate by the enzyme has been proposed. The amino acid residues Asn98 and Tyr45 are shown to form the subsites for the phosphate and the base respectively on the 5'-side of the guanine occupying the primary binding site. These studies also provide a stereochemical explanation for the specificity of the 1N subsite for adenine.     

Coenzyme specificity in aldehyde dehydrogenase

Influences on coenzyme preference are explored. Lysine 137 (192 in class 1/2 ALDH) lies close to the adenine ribose, directly interacting with the adenine ribose in NAD-specific ALDHs and the 2'-phosphate of NADP in NADP-specific ALDHs. Lys-137 in class 3 ALDH interacts with the adenine ribose indirectly through an intervening water molecule. However, this residue is present in all ALDHs and, as a result, is unlikely to directly influence coenzyme specificity. Glutamate 140 (195) coordinates the 2'- and 3'-hydroxyls of the adenine ribose of NAD in the class 3 tertiary structure. Thus, it appeared that this residue would influence coenzyme specificity. Mutation to aspartate, asparagine, glutamine or threonine shifts the coenzyme specificity towards NADP, but did not completely change the specificity. Still, the mutants show the 2'-phosphate of NADP is repelled by Glu-140 (195). Although Glu-140 (195) has a major influence on coenzyme specificity, it is not the only influence since class 3 ALDHs, can use both coenzymes, and class 2 ALDHs, which are NAD-specific, have a glutamate at this position. One explanation may be that the larger space between Lys-137 (192) and the adenine ribose hydroxyls in the class 3 ALDH:NAD binary structure may provide space to accommodate the 2'-phosphate of NADP. Also, a structural shift upon binding NADP may also occur in class 3 ALDHs to help accommodate the 2'-phosphate of NADP.     

Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques

We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans

Gox2253 from Gluconobacter oxydans belongs to the short‐chain dehydrogenases/reductases family, and catalyzes the reduction of heptanal, octanal, nonanal, and decanal with NADPH. To develop a robust working platform to engineer novel G. oxydans oxidoreductases with designed coenzyme preference, we adopted a structure based rational design strategy using computational predictions that considers the number of hydrogen bonds formed between enzyme and docked coenzyme. We report the crystal structure of Gox2253 at 2.6 Å resolution, ternary models of Gox2253 mutants in complex with NADH/short‐chain aldehydes, and propose a structural mechanism of substrate selection. Molecular dynamics simulation shows that hydrogen bonds could form between 2′‐hydroxyl group in the adenosine moiety of NADH and the side chain of Gox2253 mutant after arginine at position 42 is replaced with tyrosine or lysine. Consistent with the molecular dynamics prediction, Gox2253‐R42Y/K mutants can use both NADH and NADPH as a coenzyme. Hence, the strategies here could provide a practical platform to engineer coenzyme selectivity for any given oxidoreductase and could serve as an additional consideration to engineer substrate‐binding pockets. Proteins 2014; 82:2925–2935. © 2014 Wiley Periodicals, Inc.     

A reductase/isomerase subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) carries an NADPH and is involved in the biogenesis of the complex.

Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase, or complex I. The bacterial complex I consists of 14 subunits, whereas the mitochondrial complex contains some 25 extra subunits in addition to the homologues of the bacterial subunits. One of these extra subunits with a molecular mass of 40 kDa belongs to a heterogeneous family of reductases/isomerases with a conserved nucleotide binding site. We deleted this subunit in Neurospora crassa by gene disruption. In the mutant nuo 40, a complex I lacking the 40 kDa subunit is assembled. The mutant complex I does not contain tightly bound NADPH present in wild-type complex I. This NADPH cofactor is not connected to the respiratory electron pathway of complex I. The mutant complex has normal NADH dehydrogenase activity and contains the redox groups known for wild-type complex I, one flavin mononucleotide and four iron-sulfur clusters detectable by electron paramagnetic resonance spectroscopy. In the mutant complex these groups are all readily reduced by NADH. However, the mutant complex is not capable of reducing ubiquinone. A recently described redox group identified in wild-type complex I by UV-visible spectroscopy is not detectable in the mutant complex. We propose that the reductase/isomerase subunit with its NADPH cofactor takes part in the biosynthesis of this new redox group.     

Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity

The two-component enzyme, 4-hydroxyphenylacetate 3-monooxygenase, catalyzes the conversion of 4-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. In the overall reaction, the oxygenase component (HpaB) introduces a hydroxyl group into the benzene ring of 4-hydroxyphenylacetate using molecular oxygen and reduced flavin, while the reductase component (HpaC) provides free reduced flavins for HpaB. The crystal structures of HpaC from Thermus thermophilus HB8 in the ligand-free form, the FAD-containing form, and the ternary complex with FAD and NAD(+) were determined. In the ligand-free form, two large grooves are present at the dimer interface, and are occupied by water molecules. A structural analysis of HpaC containing FAD revealed that FAD has a low occupancy, indicating that it is not tightly bound to HpaC. This was further confirmed in flavin dissociation experiments, showing that FAD can be released from HpaC. The structure of the ternary complex revealed that FAD and NAD(+) are bound in the groove in the extended and folded conformation, respectively. The nicotinamide ring of NAD(+) is sandwiched between the adenine ring of NAD(+) and the isoalloxazine ring of FAD. The distance between N5 of the isoalloxazine ring and C4 of the nicotinamide ring is about 3.3 A, sufficient to permit hydride transfer. The structures of these three states are essentially identical, however, the side chains of several residues show small conformational changes, indicating an induced fit upon binding of NADH. Inactivity with respect to NADPH can be explained as instability of the binding of NADPH with the negatively charged 2'-phosphate group buried inside the complex, as well as a possible repulsive effect by the dipole of helix alpha1. A comparison of the binding mode of FAD with that in PheA2 from Bacillus thermoglucosidasius A7, which contains FAD as a prosthetic group, reveals remarkable conformational differences in a less conserved loop region (Gly83-Gly94) involved in the binding of the AMP moiety of FAD. These data suggest that variations in the affinities for FAD in the reductases of the two-component flavin-diffusible monooxygenase family may be attributed to difference in the interaction between the AMP moiety of FAD and the less conserved loop region which possibly shows structural divergence.