NADPH-binding Domain Research Articles

Conformational flexibility ofMycobacterium tuberculosisthioredoxin reductase: crystal structure and normal-mode analysis

The thioredoxin system exists ubiquitously and participates in essential antioxidant and redox-regulation processes via a pair of conserved cysteine residues. In Mycobacterium tuberculosis, which lacks a genuine glutathione system, the thioredoxin system provides reducing equivalents inside the cell. The three-dimensional structure of thioredoxin reductase from M. tuberculosis has been determined at 3 A resolution. TLS refinement reveals a large libration axis, showing that NADPH-binding domain has large anisotropic disorder. The relative rotation of the NADPH domain with respect to the FAD domain is necessary for the thioredoxin reduction cycle, as it brings the spatially distant reacting sites close together. Normal-mode analysis carried out based on the elastic network model shows that the motion required to bring about the functional conformational change can be accounted for by motion along one single mode. TLS refinement and normal-mode analysis thus enhance our understanding of the associated conformational changes.

Ablation of the otcC Gene Encoding a Post-polyketide Hydroxylase from the Oxytetracyline Biosynthetic Pathway in Streptomyces rimosus Results in Novel Polyketides with Altered Chain Length

Oxytetracycline (OTC) is a 19-carbon polyketide antibiotic made by Streptomyces rimosus. The otcC gene encodes an anhydrotetracycline oxygenase that catalyzes a hydroxylation of the anthracycline structure at position C-6 after biosynthesis of the polyketide backbone is completed. A recombinant strain of S. rimosus that was disrupted in the genomic copy of otcC synthesized a novel C-17 polyketide. This result indicates that the absence of the otcC gene product significantly influences the ability of the OTC "minimal" polyketide synthase to make a polyketide product of the correct chain length. A mutant copy of otcC was made by site-directed mutagenesis of three essential glycine codons located within the putative NADPH-binding domain. The mutant gene was expressed in Escherichia coli, and biochemical analysis confirmed that the gene product was catalytically inactive. When the mutant gene replaced the ablated gene in the chromosome of S. rimosus, the ability to make a 19-carbon backbone was restored, indicating that OtcC is an essential partner in the quaternary structure of the synthase complex.

Crystal Structure of a Novel FAD-, FMN-, and ATP-containing l-Proline Dehydrogenase Complex from Pyrococcus horikoshii

Two novel types of dye-linked L-proline dehydrogenase complex (PDH1 and PDH2) were found in a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Here we report the first crystal structure of PDH1, which is a heterooctameric complex (alphabeta)4 containing three different cofactors: FAD, FMN, and ATP. The structure was determined by x-ray crystallography to a resolution of 2.86 angstroms. The structure of the beta subunit, which is an L-proline dehydrogenase catalytic component containing FAD as a cofactor, was similar to that of monomeric sarcosine oxidase. On the other hand, the alpha subunit possessed a unique structure composed of a classical dinucleotide fold domain with ATP, a central domain, an N-terminal domain, and a Cys-clustered domain. Serving as a third cofactor, FMN was located at the interface between the alpha and beta subunits in a novel configuration. The observed structure suggests that FAD and FMN are incorporated into an electron transfer system, with electrons passing from the former to the latter. The function of ATP is unknown, but it may play a regulatory role. Although the structure of the alpha subunit differs from that of the beta subunit, except for the presence of an analogous dinucleotide domain with a different cofactor, the structural characteristics of PDH1 suggest that each represents a divergent enzyme that arose from a common ancestral flavoenzyme and that they eventually formed a complex to gain a new function. The structural characteristics described here reveal the PDH1 complex to be a unique diflavin dehydrogenase containing a novel electron transfer system.

X-ray Structures of NADPH-dependent Carbonyl Reductase from Sporobolomyces salmonicolor Provide Insights into Stereoselective Reductions of Carbonyl Compounds

The X-ray structures of red yeast Sporobolomyces salmonicolor carbonyl reductase (SSCR) and its complex with a coenzyme, NADPH, have been determined at a resolution of 1.8A and 1.6A, respectively. SSCR was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=54.86 A, b=83.49 A, and c=148.72 A. On its cocrystallization with NADPH, isomorphous crystals of the SSCR/NADPH complex were obtained. The structure of SSCR was solved by a single wavelength anomalous diffraction measurement using a selenomethionine-substituted enzyme, and that of the SSCR/NADPH complex was solved by a molecular replacement method using the solved structure of SSCR. The structures of SSCR and the SSCR/NADPH complex were refined to an R-factor of 0.193 (R(free)=0.233) and 0.211 (R(free)=0.238), respectively. SSCR has two domains, an NADPH-binding domain and a substrate-binding domain, and belongs to the short-chain dehydrogenases/reductases family. The structure of the NADPH-binding domain and the interaction between the enzyme and NADPH are very similar to those found in other structure-solved enzymes belonging to the short-chain dehydrogenases/reductases family, while the structure of the substrate-binding domain is unique. SSCR has stereoselectivity in its catalytic reaction, giving rise to excessive production of (S)-alcohols from ethyl 4-chloro-3-oxobutanoate. The X-ray structure of the SSCR/NADPH complex and preliminary modeling show that the formation of the hydrophobic channel induced by the binding of NADPH is closely related to the stereoselective reduction by SSCR.

Human neuronal nitric oxide synthase can catalyze one-electron reduction of adriamycin: role of flavin domain

We have analyzed the mechanism of one-electron reduction of adriamycin (Adr) using recombinant full-length human neuronal nitric-oxide synthase and its flavin domains. Both enzymes catalyzed aerobic NADPH oxidation in the presence of Adr. Calcium/calmodulin (Ca 2+/CaM) stimulated the NADPH oxidation of Adr. In the presence or absence of Ca 2+/CaM, the flavin semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by Adr. The FAD–NADPH binding domain did not significantly catalyze the reduction of Adr. Neither the FAD semiquinone (FADH ) nor the air-stable semiquinone (FAD–FMNH ) reacted rapidly with Adr. These data indicate that the fully reduced species of FMN (FMNH 2) donates one electron to Adr, and that the rate of Adr reduction is stimulated by a rapid electron exchange between the two flavins in the presence of Ca 2+/CaM. Based on these findings, we propose a role for the FAD–FMN pair in the one-electron reduction of Adr.

Crystal Structures of Pinoresinol-Lariciresinol and Phenylcoumaran Benzylic Ether Reductases and Their Relationship to Isoflavone Reductases

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

Crystal structure of 1-deoxy- d-xylulose-5-phosphate reductoisomerase from Zymomonas mobilis at 1.9-Å resolution

1-Deoxy- d-xylulose-5-phosphate reductoisomerase (DXR) is the second enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. The structure of the apo-form of this enzyme from Zymomonas mobilis has been solved and refined to 1.9-Å resolution, and that of a binary complex with the co-substrate NADPH to 2.7-Å resolution. The subunit of DXR consists of three domains. Residues 1–150 form the NADPH binding domain, which is a variant of the typical dinucleotide-binding fold. The second domain comprises a four-stranded mixed β-sheet, with three helices flanking the sheet. Most of the putative active site residues are located on this domain. The C-terminal domain (residues 300–386) folds into a four-helix bundle. In solution and in the crystal, the enzyme forms a homo-dimer. The interface between the two monomers is formed predominantly by extension of the sheet in the second domain. The adenosine phosphate moiety of NADPH binds to the nucleotide-binding fold in the canonical way. The adenine ring interacts with the loop after β1 and with the loops between α2 and β2 and α5 and β5. The nicotinamide ring is disordered in crystals of this binary complex. Comparisons to Escherichia coli DXR show that the two enzymes are very similar in structure, and that the active site architecture is highly conserved. However, there are differences in the recognition of the adenine ring of NADPH in the two enzymes.

Subunit and cofactor binding of Saccharomyces cerevisiae sulfite reductase - towards developing wine yeast with lowered ability to produce hydrogen sulfide

Wine yeast, in common with other industrial Saccharomyces cerevisiae yeast, can produce hydrogen sulfide during alcoholic fermentation. The aim of this work was to evaluate a genetic strategy for reducing hydrogen sulfide production by lowering the activity of NADPH-dependent sulfite reductase, a key enzyme in the biosynthesis of the sulfur-containing amino acids. The role of amino acids predicted to be necessary for cofactor binding was investigated by comparisons with other proteins in the ferredoxin family. Substitutions in the glycine-rich loop domain (glycines 891 and 893) and at serine 953 of the alpha subunit, Met10p, appear to strongly affect NADPH binding. However, mutations in two amino acids that are completely conserved within the NADPH binding domain of the ferredoxin reductase family, lysine 959 and cysteine 987, have minimal effect on enzyme activity. Serine 820 is indicated to be essential for efficient FAD binding. The two-hybrid system was used to confirm that Met 1 0p interacts with Met5p, the predicted beta subunit of sulfite reductase. Binding of the subunits could only be demonstrated in the absence of methionine, indicating that an additional factor, possibly siroheme, is required to mediate this interaction. Strategies to decrease, but not eliminate, sulfite reductase activity in the cell using identified mutants were tested, and the findings are discussed with the a view to producing improved wine strains.

The crystal structure of shikimate dehydrogenase (AroE) reveals a unique NADPH binding mode.

Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-A resolution and its complex with NADPH to 1.95-A resolution. The molecule contains two domains, a catalytic domain with a novel open twisted alpha/beta motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.

A novel flavin-containing monooxygenase from Methylophaga sp. strain SK1 and its indigo synthesis in Escherichia coli

We cloned a gene from Methylophaga sp. strain SK1. This gene was responsible for producing, the blue pigment, indigo. The complete open reading frame was 1371 bp long, which encodes a protein of 456 amino acids. The molecular mass of the encoded protein was 105 kDa, consisting of homodimer of 54 kDa with an isoelectric point of 5.14. The deduced amino acid sequence from the gene showed approximately 30% identities with flavin-containing monooxygenases (FMOs) of human (FMO1–FMO5), Arabidopsis, and yeast. It contained three characteristic sequence motifs of FMOs: FAD binding domain, FMO-identifying sequence motif, and NADPH binding domain. In addition, the biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, we assigned the enzyme to be a bacterial FMO. The recombinant Escherichia coli expressing the bacterial FMO produced up to 160 mg of indigo per liter in the tryptophan medium after 12 h cultivation. This suggests that the recombinant strain has a potential to be applied in microbial indigo production.

Characterization of thioredoxin reductase genes ( trr1) from Pneumocystis carinii and Pneumocystis jiroveci

We have characterized the thioredoxin reductase ( trr1) genes from Pneumocystis carinii and Pneumocystis jiroveci, and have demonstrated that multiple copies of an ∼500 base pair fragment of the trr1 gene are present in P. carinii, but not in P. jiroveci. Thioredoxin reductases encoded by the full-length genes have predicted molecular weights of ∼35,000 and show high homology to yeast Trr1. An NADPH-binding domain with a putative redox active site CAVC as well as an flavin-adenine dinucleotide-binding domain are highly conserved in both proteins, which were 85% identical. The multicopy trr1 gene fragments in P. carinii are not transcribed or expressed. Duplication of the gene fragment likely occurred in conjunction with duplication of the kexin homologue, protease-1, which is located immediately upstream of the trr1 gene. Thioredoxin reductase, an enzyme implicated in the growth, survival and pathogenicity of certain microbes, could be a potential target for therapeutic intervention in Pneumocystis infection.

Site-directed mutational analysis of the novel catalytic domains of alpha-aminoadipate reductase (Lys2p) from Candida albicans.

The alpha-aminoadipate reductase, a novel enzyme in the alpha-aminoadipic acid pathway for the biosynthesis of lysine in fungi, catalyzes the conversion of alpha-aminoadipic acid to alpha-aminoadipic-delta-semialdehyde in the presence of ATP, NADPH and MgCl(2). This reaction requires two distinct gene products, Lys2p and Lys5p. In the presence of CoA, Lys5p posttranslationally activates Lys2p for the alpha-aminoadipate reductase activity. Sequence alignments indicate the presence of all functional domains required for the activation, adenylation, dehydrogenation and alpha-aminoadipic acid binding in the Lys2p. In this report we present the results of site-directed mutational analysis of the conserved amino acid residues in the catalytic domains of Lys2p from the pathogenic yeast Candida albicans. Mutants were generated in the LYS2 sequence of pCaLYS2SEI by PCR mutagenesis and expressed in E. coli BL21 cells. Recombinant mutants and the wild-type Lys2p were analyzed for their alpha-aminoadipate reductase activity. Substitution of threonine 416, glycine 418, serine 419, and lysine 424 of the adenylation domain (TXGSXXXXK, residues 416-424) resulted in a significant reduction in alpha-aminoadipate reductase activity compared to the unmutagenized Lys2p control. Similarly replacement of glycine 978, threonine 980, glycine 981, phenylalanine 982, leucine 983 and glycine 984 of the NADPH binding domain (GXTGFLG, residues 978-984) caused a drastic decrease in alpha-aminoadipate reductase activity. Finally, substitution of histidine 460, aspartic acid 461, proline 462, isoleucine 463, glutamine 464, arginine 465, and aspartic acid 466 of the putative alpha-aminoadipic acid binding domain (HDPIQRD, residues 460-466) resulted in a highly reduced alpha-aminoadipate reductase activity. These results confirm the hypothesis that specific amino acid residues in highly conserved catalytic domains of Lys2p are essential for the alpha-aminoadipate reductase activity.

Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase-thioredoxin h system from wheat.

Thioredoxins h are ubiquitous proteins reduced by NADPH- thioredoxin reductase (NTR). They are able to reduce disulphides in target proteins. In monocots, thioredoxins h accumulate at high level in seeds and show a predominant localization in the nucleus of seed cells. These results suggest that the NTR-thioredoxin h system probably plays an important role in seed physiology. To date, the study of this system in monocots is limited by the lack of information about NTR. In the present study, we describe the cloning of a full-length cDNA encoding NTR from wheat ( Triticum aestivum ). The polypeptide deduced from this cDNA shows close similarity to NTRs from Arabidopsis, contains FAD- and NADPH-binding domains and a disulphide probably interacting with the disulphide at the active site of thioredoxin h. Wheat NTR was expressed in Escherichia coli as a His-tagged protein. The absorption spectrum of the purified recombinant protein is typical of flavoenzymes. Furthermore, it showed NADPH-dependent thioredoxin h reduction activity, thus confirming that the cDNA clone reported in the present study encodes wheat NTR. Using the His-tagged NTR and TRXhA (wheat thioredoxin h ), we successfully reconstituted the wheat NTR-thioredoxin h system in vitro, as shown by the insulin reduction assay. A polyclonal antibody was raised against wheat NTR after immunization of rabbits with the purified His-tagged protein. This antibody efficiently detected a single polypeptide of the corresponding molecular mass in seed extracts and it allowed the analysis of the pattern of accumulation of NTR in different wheat organs and developmental stages. NTR shows a wide distribution in wheat, but, surprisingly, its accumulation in seeds is low, in contrast with the level of thioredoxins h.

Identification and heterologous expression of the cytochrome P450 oxidoreductase from the white-rot basidiomycete Coriolus versicolor.

A cDNA encoding cytochrome P450 oxidoreductase (CPR) from the lignin-degrading basidiomycete Coriolus versicolor was identified using RT-PCR. The full-length cDNA consisted of 2,484 nucleotides with a poly(A) tail, and contained an open reading frame. The G+C content of the cDNA isolated was 60%. A deduced protein contained 730 amino acid residues with a calculated molecular weight of 80.7 kDa. The conserved amino acid residues involved in functional domains such as FAD-, FMN-, and NADPH-binding domains, were all found in the deduced protein. A phylogenetic analysis demonstrated that C. versicolor CPR is significantly similar to CPR of the basidiomycete Phanerochaete chrysosporium and that they share the same major branch in the fungal cluster. A recombinant CPR protein was expressed using a pET/ Escherichia coli system. The recombinant CPR protein migrated at 81 kDa on SDS polyacrylamide gel electrophoresis. It exhibited an NADPH-dependent cytochrome c reducing activity.

Mutational Analysis of Human Thioredoxin Reductase 1: EFFECTS ON p53-MEDIATED GENE EXPRESSION AND INTERFERON AND RETINOIC ACID-INDUCED CELL DEATH

The interferon (IFN)-beta and all-trans-retinoic acid combination suppresses tumor growth by inducing apoptosis in several tumor cell lines. A genetic technique permitted the isolation of human thioredoxin reductase (TR) as a critical regulator of IFN/all-trans-retinoic acid-induced cell death. Our recent studies have shown that TR1:thioredoxin 1-regulated cell death is effected in part through the activation of p53-dependent responses. To understand its death regulatory function, we have performed a mutational analysis of TR. Human TR1 has three major structural domains, the FAD binding domain, the NADPH binding domain, and an interface domain (ID). Here, we show that the deletion of the C-terminal interface domain results in a constitutive activation of TR-dependent death responses and promotes p53-dependent gene expression. TR mutant without the ID still retains its dependence on thioredoxin for promoting these responses. Thus, our data suggest that TR-ID acts as a regulatory domain.

Flavin photochemistry in the analysis of electron transfer reactions: role of charged and hydrophobic residues at the carboxyl terminus of ferredoxin–NADP + reductase in the interaction with its substrates

The enzyme Ferredoxin-NADP + reductase participates in the reductive side of the photosynthetic chain transferring electrons from reduced Ferredoxin (Fd) (or Flavodoxin (Fld)) to NADP +, a process that yields NADPH that can be used in many biosynthetic dark reactions. The involvement of specific amino acids in the interaction between the two proteins has been studied using site-directed mutagenesis. In the present study, the participation of charged (H299), polar (T302) or hydrophobic (V300) amino acid residues that are in the NADP +-binding domain of the reductase have been examined by analyzing its C-terminal region, which is located close to the active site. Stopped-flow and laser flash photolysis results of the reaction in which these mutant proteins participate show very little differences with respect to the wild-type protein. These results suggest that the NADPH-binding domain of the reductase has little effect on the processes of recognition and electron transfer to (and from) Fd or Fld, according to the recently reported crystallographic structure of the FNR/Fd complex

Crystal Structure of the FAD/NADPH-binding Domain of Rat Neuronal Nitric-oxide Synthase: COMPARISONS WITH NADPH-CYTOCHROME P450 OXIDOREDUCTASE

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP(+), has been determined at 1.9 A resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP(+) binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.

Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH.

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.

Hydrophobicity of the NADPH binding domain of camel lens ζ-crystallin

Interaction of camel lens ζ-crystallin with the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) enhanced the ANS fluorescence and quenched the protein fluorescence. Both of these events were concentration-dependent and showed typical saturation curves suggesting specific ANS-ζ-crystallin binding. Quantitative analysis indicated that 1 mole ζ-crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace ζ-crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in ζ-crystallin, possibly at the NADPH binding site. α-Crystallin as well as NADPH protected ζ-crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH:quinone oxidoreductase activity of ζ-crystallin was inhibited by ANS with NADPH as electron donor and PQ as electron acceptor. Lineweaver-Burk plots indicated mixed-type inhibition with respect to NADPH, with a K i of 2.3 μM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant ( K′ I) of 12 μM for the ζ-crystallin-NADPH-ANS complex. The K i being smaller than K′ I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-ζ-crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time.

Crystal Structure of Saccharopine Reductase from Magnaporthe grisea, an Enzyme of the α-Aminoadipate Pathway of Lysine Biosynthesis

Background: The biosynthesis of the essential amino acid lysine in higher fungi and cyanobacteria occurs via the α-aminoadipate pathway, which is completely different from the lysine biosynthetic pathway found in plants and bacteria. The penultimate reaction in the α-aminoadipate pathway is catalysed by NADPH-dependent saccharopine reductase. We set out to determine the structure of this enzyme as a first step in exploring the structural biology of fungal lysine biosynthesis. Results: We have determined the three-dimensional structure of saccharopine reductase from the plant pathogen Magnaporthe grisea in its apo form to 2.0 Å resolution and as a ternary complex with NADPH and saccharopine to 2.1 Å resolution. Saccharopine reductase is a homodimer, and each subunit consists of three domains, which are not consecutive in amino acid sequence. Domain I contains a variant of the Rossmann fold that binds NADPH. Domain II folds into a mixed seven-stranded β sheet flanked by α helices and is involved in substrate binding and dimer formation. Domain III is all-helical. The structure analysis of the ternary complex reveals a large movement of domain III upon ligand binding. The active site is positioned in a cleft between the NADPH-binding domain and the second α/β domain. Saccharopine is tightly bound to the enzyme via a number of hydrogen bonds to invariant amino acid residues. Conclusions: On the basis of the structure of the ternary complex of saccharopine reductase, an enzymatic mechanism is proposed that includes the formation of a Schiff base as a key intermediate. Despite the lack of overall sequence homology, the fold of saccharopine reductase is similar to that observed in some enzymes of the diaminopimelate pathway of lysine biosynthesis in bacteria. These structural similarities suggest an evolutionary relationship between two different major families of amino acid biosynthetic pathway, the glutamate and aspartate families.