Characterization of dihydrodipicolinate reductase from Thermotoga maritima reveals evolution of substrate binding kinetics

In lysine biosynthesis, dihydrodipicolinate reductase (DHDPR) catalyses the formation of tetrahydrodipicolinate. Unlike DHDPR enzymes from Escherichia coli and Mycobacterium tuberculosis, which have dual specificity for both NADH and NADPH as co-factors, the enzyme from Thermotoga maritima has a significantly greater affinity for NADPH. Despite low sequence identity with the E. coli and M. tuberculosis DHDPR enzymes, DHDPR from T. maritima has a similar catalytic site, with many conserved residues involved in interactions with substrates. This suggests that as the enzyme evolved, the co-factor specificity was relaxed. Kinetic studies show that the T. maritima DHDPR enzyme is inhibited by high concentrations of its substrate, DHDP, and that at high concentrations NADH also acts as an inhibitor of the enzyme, suggesting a novel method of regulation for the lysine biosynthetic pathway. Increased thermal stability of the T. maritima DHDPR enzyme may be associated with the lack of C-terminal and N-terminal loops that are present in the E. coli DHDPR enzyme.     

Structure and nucleotide specificity of Staphylococcus aureus dihydrodipicolinate reductase (DapB)

Lysine biosynthesis proceeds by the nucleotide-dependent reduction of dihydrodipicolinate (DHDP) to tetrahydrodipicolinate (THDP) by dihydrodipicolinate reductase (DHDPR). The S. aureus DHDPR structure reveals different conformational states of this enzyme even in the absence of a substrate or nucleotide-cofactor. Despite lacking a conserved basic residue essential for NADPH interaction, S. aureus DHDPR differs from other homologues as NADPH is a more preferred co-factor than NADH. The structure provides a rationale-Lys35 compensates for the co-factor site mutation. These observations are significant for bi-ligand inhibitor design that relies on ligand-induced conformational changes as well as co-factor specificity for this important drug target.     

Cooperative interaction of reduced pyridine nucleotides with estrogen synthetase of human placental microsomes

The estrogen synthetase present in human placental microsomes appears to be dependent on the cooperative interaction of the reduced cofactors NADPH and NADH for optimal activity. Using steady-state concentrations of either cofactor, it was found that while the estrogen synthetase activity followed hyperbolic saturation kinetics with NADPH (K_mapp = 14 μM), the enzyme followed sigmoidal saturation kinetics when the cofactor was NADH, with the half-maximum velocity attained at a cofactor concentration of 1.1 mm. The maximum velocity obtained with NADPH as the cofactor was greater than with corresponding concentrations of NADH. Estrogen synthetase activity in the presence of NADH was not due to NADPH contamination. NADH, in the presence of small concentrations of NADPH (0.5 to 5 μm), stimulated significantly the rate of estrogen formation from androstenedione by placental microsomes and, in addition, the enzyme saturation kinetics changed from sigmoidal to hyperbolic, thus mimicking the effect of NADPH. Estrogen synthetase activity, measured in the presence of 1 mm NADH, was stimulated in a dose-dependent manner by NADPH (K_mapp = 0.4 μM NADPH) and, when the enzyme was measured in the presence of 5 μm NADPH, the activity was stimulated in a dose-dependent manner by NADH (K_mapp = 45 μM NADH). Estrogen synthetase activity measured in the presence of NADH, without and with NADPH (1 μm) remained linear both with time of incubation for approximately 15 min and with microsomal protein concentration up to 3 mg/ml. The apparent K_m of estrogen synthetase for androstenedione, when measured in the presence of NADH, was 1 μm. The synergistic interaction between NADH and NADPH in stimulating placental estrogen synthetase activity observed in vitro may, conceivably, take place in vivo in the intact placenta.     

Comparative structure and function analyses of native and his-tagged forms of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Given the rise of multi drug resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), there is an urgent need to discover new antimicrobial agents. A validated but as yet unexplored target for new antibiotics is dihydrodipicolinate reductase (DHDPR), an enzyme that catalyzes the second step of the lysine biosynthesis pathway in bacteria. We report here the cloning, expression and purification of N-terminally his-tagged recombinant DHDPR from MRSA (6H-MRSA-DHDPR) and compare its secondary and quaternary structure with the wild type (MRSA-DHDPR) enzyme. Comparative analyses demonstrate that recombinant 6H-MRSA-DHDPR is folded and adopts the native tetrameric quaternary structure in solution. Furthermore, kinetic studies show 6H-MRSA-DHDPR is functional, displaying parameters for K(m)(NADH) of 6.0μM, K(m)(DHDP) of 22μM, and k(cat) of 21s(-1), which are similar to those reported for the native enzyme. The solution properties and stability of the 6H-MRSA-DHDPR enzyme are also reported in varying physicochemical conditions.     

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.     

Identification of nitric oxide and nitrous oxide as products of nitrite reduction by Pseudomonas cytochrome oxidase (nitrate reductase)

The cytosol fraction of rat adrenocortical tissue contains comparatively high levels of two prostaglandin metabolizing enzymes. The first, prostaglandin-9-ketoreductase, utilizes NADPH more effectively than NADH as cofactor, is inhibited by NADP, and exhibits an apparent K_m of 304 μM for PGE₁. 15-hydroxyprostaglandin dehydrogenase, tentatively identified as the type II NADP-dependent isozyme, is inhibited by NADPH but not NADH, and exhibits an apparent K_m of 157 μM when PGE₁ is used as substrate. Changes in specific activities of the two enzymes following ACTH, hypophysectomy, or dexamethasone treatment are inconclusive in defining a chronic regulatory role for adrenocorticotropin.     

Purification and partial characterization of an NADH-linked delta13-15-ketoprostaglandin reductase from human placenta.

A Δ¹³-15-ketoprostaglandin reductase has been isolated from human placenta and purified 800-fold. The enzyme utilizes NADH as a cofactor but not NADPH. It reduces the 13,14 double bond in 15-ketoprostaglandin E₁, E₂ and F_2α. The K_M apparent for NADH is 54.8 μM and the K_M apparent for 15-ketoprostaglanding E₂ is 7.0 μM. The partially purified enzyme contains no 15-hydroxyprostaglandin dehydrogenase activity.     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.     

A novel substrate for yeast alcohol dehydrogenase – p-nitroso-N,N-dimethylaniline

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzes the novel reduction of p-nitro-so-N,N-dimethylaniline with NADH as a cofactor. Apparent kinetic constants for this enzymatic reaction are: V ₂=2.1 s^–1, K _Q=456 M, K _iQ=119 M, and K _P=1.47 mM, at pH 8.9, 25 °C. This reaction is especially useful for the quantitative determination of NAD⁺ and NADH by enzymatic cycling.     

Genetic changes that increase 5-hydroxymethyl furfural resistance in ethanol-producing <Emphasis Type="Italic">Escherichia coli</Emphasis> LY180

The ability of a biocatalyst to tolerate furan inhibitors present in hemicellulose hydrolysates is important for the production of renewable chemicals. This study shows EMFR9, a furfural-tolerant mutant of ethanologenic E. coli LY180, has also acquired tolerance to 5-hydroxymethyl furfural (5-HMF). The mechanism of action of 5-HMF and furfural appear similar. Furan tolerance results primarily from lower expression of yqhD and dkgA, two furan reductases with a low K_m for NADPH. Furan tolerance was also increased by adding plasmids encoding a NADPH/NADH transhydrogenase (pntAB). Together, these results support the hypothesis that the NADPH-dependent reduction of furans by YqhD and DkgA inhibits growth by competing with biosynthesis for this limiting cofactor.     

Heterologous expression,purification, and characterization of xylose reductase from <Emphasis Type="Italic">Candida shehatae</Emphasis>

The xylose reductase (XR) gene (xyl1) from Candida shehatae was cloned and expressed in Escherichia coli, and purified as a His₆-tagged fusion protein. The recombinant XR had K_m values for NADH than NADPH of 150 μM and 20 μM, respectively. The optimal reaction was at pH 6.5 and 35°C. The enzyme was specific for d-xylese.     

Pyridine nucleotide specificity and other properties of purified nitrate reductase from Chlorella variegata

Nitrate reductase (NR) (EC 1.6.6.2) from Chlorella variegata 211/10d has been purified by blue sepharose affinity chromatography. The enzyme can utilise NADH or NADPH for nitrate reduction with apparent K _m values of 11.5 M and 14.5 M, respectively. Apparent K _m values for nitrate are 0.13 mM (NADH-NR) and 0.14 mM (NADPH-NR). The diaphorase activity of the enzyme is inhibited strongly by parachloromercuribenzoic acid; NADH or NADPH protects the enzyme against this inhibition. NR proper activity of the enzyme is partially inactive after extraction and may be activated after the addition of ferricyanide. The addition of NAD(P)H and cyanide causes a reversible inactivation of the NR proper activity although preincubation with either NADH or NADH and ADP has no significant effect.Abbreviations NR Nitrate reductase - FAD Flavin-adenine dinucleotide - FMN Riboflavin 5-phosphate - p-CMB para-Chloromercuribenzoic - BV Benzyl viologen     

Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis

Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine–synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T _0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-β -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.     

Reduced Pyridine Nucleotide Oxidases of Euglena gracilis var. bacillaris*

SYNOPSIS. Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (K_m) determined were as follows: NADH diaphorase, 350 μM; NADPH diaphorase, 200 μM; NADH cytochrome c reductase, 13 μM; NADPH cytochrome c reductase, 9 μM; NADH oxidase, 100 μM; NADPH oxidase 150 μM; NADH lipoyl dehydrogenase, 0.35 μM. Enzyme activities after storage at –5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

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

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

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K_d, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC₅₀ of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and ¹⁵N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including ¹⁵N-¹H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.     

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

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

Glutathione increases the binding affinity of a bovine B12 trafficking chaperone bCblC for vitamin B12

Intracellular B₁₂ metabolism involves a B₁₂ trafficking chaperone CblC that is well conserved in mammals including human. The protein CblC is known to bind cyanocobalamin (CNCbl, vitamin B₁₂) inducing the base-off transition and convert it into an intermediate that can be used in enzyme cofactor synthesis. The binding affinity of human CblC for CNCbl was determined to be K_d = ≈6–16 μM, which is relatively low considering sub-micromolar B₁₂ concentrations (0.03–0.7 μM) in normal cells. In the current study, we discovered that the base-off transition of CNCbl upon binding to bCblC, a bovine homolog of human CblC, is facilitated in the presence of reduced form of glutathione (GSH). In addition, GSH dramatically increases the binding affinity for CNCbl lowering the K_d from 27.1 ± 0.2–0.24 ± 0.09 μM. The effect of GSH is due to conformational change of bCblC upon binding with GSH, which was indicated by limited proteolysis and urea-induced equilibrium denaturation of the protein. The results of this study suggest that GSH positively modulates bCblC by increasing the binding affinity for CNCbl, which would enhance functional efficiency of the protein.     

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K_m values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K_i value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.     

Compartmentation and Activities of Enzymes Involved in the Metabolism of Amino Acids Implicated in Osmoregulatory Mechanisms in Acanthamoeba castellanii1

Glutamate dehydrogenase in Acanthamoeba castellanii is an NAD-dependent cytosolic enzyme. This is similar to glutamate dehydrogenases in Phycomycetes, but very different from the dual coenzyme-specific enzymes located in mitochondria in animals and in mitochondria and chloroplasts in higher plants. Pyrroline-5-carboxylate (P-5-C) reductase occurs also in the cytoplasm in A. castellanii and has very high affinities for L-P-5-C (K_m= 12 μM) and NADH (K_m= 15 _μM). In contrast, ornithine aminotransferase and proline oxidase are mitochondrial enzymes. No proline-inhibited γ-glutamyl kinase was detected while an active glutamine synthetase was found in the cytosolic compartment. Evidence for a mitochondrial transport system for L-proline was obtained. Two possible pathways for proline biosynthesis in A. castellanii are discussed based on information obtained about activities and subcellular compartmentation of enzymes.