Evidence for expression of 11β-hydroxysteroid dehydrogenase type3 (HSD11B3/HSD11B1L) in neonatal pig testis

11β-hydroxysteroid dehydrogenase (HSD11B) catalyzes the interconversion between active and inactive glucocorticoid, and is known to exist as two distinct isozymes: HSD11B1 and HSD11B2. A third HSD11B isozyme, HSD11B1L (SCDR10b), has recently been identified. Human HSD11B1L, which was characterized as a unidirectional NADP⁺-dependent cortisol dehydrogenase, appears to be specifically expressed in the brain. We previously reported that HSD11B1 and abundant HSD11B2 isozymes are expressed in neonatal pig testis and the Km for cortisol of NADP⁺-dependent dehydrogenase activity of testicular microsomes obviously differs from the same activity catalyzed by HSD11B1 from pig liver microsomes. Therefore, we hypothesized that the neonatal pig testis also expresses the third type of HSD11B isozyme, and we herein examined further evidence regarding the expression of HSD11B1L. (1) The inhibitory effects of gossypol and glycyrrhetinic acid on pig testicular microsomal NADP⁺-dependent cortisol dehydrogenase activity was clearly different from that of pig liver microsomes. (2) A highly conserved human HSD11B1L sequence was observed by RT-PCR in a pig testicular cDNA library. (3) mRNA, which contains the amplified sequence, was evaluated by real-time PCR and was most strongly expressed in pig brain, and at almost the same levels in the kidney as in the testis, but at lower levels in the liver. Based on these results, neonatal pig testis appears to express glycyrrhetinic acid-resistant HSD11B1L as a third HSD11B isozyme, and it may play a physiologically important role in cooperation with the abundantly expressed HSD11B2 isozyme in order to prevent Leydig cell apoptosis or GC-mediated suppression of testosterone production induced by high concentrations of activated GC in neonatal pig testis.     

Regulation of glucocorticoid metabolism in the boar testis and caput epididymidis by the gonadotrophin-cAMP signalling pathway

In target tissues, cortisol is metabolised by two 11β-hydroxysteroid dehydrogenase (11βHSD) isoenzymes, namely 11βHSD1 and 11βHSD2, both of which are co-expressed in the boar testis and reproductive tract. The present study has assessed whether cortisol-cortisone metabolism in boar testis and caput epididymidis can be regulated via the gonadotrophin-cAMP signalling pathway. 11βHSD activities were measured by using a radiometric conversion assay in static tissue culture. In both testis and caput epididymidis, the net reduction of cortisone but not the net oxidation of cortisol, was significantly decreased by luteinising hormone (by 53?±?20% and 45?±?9%, respectively, P?<?0.05), forskolin (by 60?±?7% and 57?±?9%, respectively, P?<?0.01) and 8-bromo-cAMP (by 54?±?4% and 64?±?1%, respectively, P?<?0.01). This suppression of 11-ketosteroid reductase activity in the boar testis by forskolin could be attenuated by the protein kinase A (PKA) inhibitor, H89. Hence, within the boar testis and the caput epididymidis, the local actions of glucocorticoids are modulated by gonadotrophin-cAMP-PKA signalling via their selective effects on the reductase activity of 11βHSD.     

Detection of 15-hydroxyprostaglandin dehydrogenase in bovine mesenteric blood vessels

Two types of 15-hydroxyprostaglandin dehydrogenase (NAD⁺ and NADP⁺ dependent) were demonstrated in bovine mesentric arteries and veins. The 15-hydroxyprostaglandin dehydrogenase activity was found in the high-speed supernatant, suggesting that these enzymes are associated with the cytoplasmic fraction of the blood vessels. The levels of activities of both NAD⁺- and NADP⁺-dependent dehydrogenases were similar in mesentric blood vessels. Prostaglandin F_2α was preferred to the prostaglandin E₂ as subtrate by both NAD⁺ and NADP⁺ dependent enzymes. The presence of 15-hydroxyprostaglandin dehydrogenase in blood vessels may play a siginificant role in the regulation of intracellular levels of prostaglandins of the E and F series in blood vessels.     

Molecular docking and structural analysis of cofactor-protein interaction between NAD⁺ and 11β-hydroxysteroid dehydrogenase type 2

Molecular docking and structural analysis of the cofactor-protein interaction between NAD⁺ and human (h) or mouse (m) 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) were performed with the molecular operating environment (MOE). 11βHSD1 (PDB code: 3HFG) was selected as a template for the 3D structure modeling of 11βHSD2. The MOE docking (MOE-dock) and the alpha sphere and excluded volume-based ligand-protein docking (ASE-dock) showed that both NAD⁺-h11βHSD2 and NAD⁺-m11βHSD2 models have a similar binding orientation to the template cofactor-protein model. Our present study also revealed that Asp91, Phe94, Tyr232 and Thr267 could be of importance in the interaction between NAD⁺ and 11βHSD2. NADP⁺ was incapable of entering into the cofactor-binding site of the 11βHSD2 models. The present study proposes the latest models for 11βHSD2 and its cofactor NAD⁺, and to the best of our knowledge, this is the first report of a m11βHSD2 model with NAD⁺.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

NAD+ -dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation

DEAE-cellulose chromatography of extracts of free-living Rhizobium meliloti cells revealed separate NAD⁺-dependent and NADP⁺-dependent malic enzyme activities. The NAD⁺ malic enzyme exhibited more activity with NAD⁺ as cofactor, but also showed some activity with NADP⁺. The NADP⁺ malic enzyme only showed activity when NADP⁺ was supplied as cofactor. Three independent transposon-induced mutants of R. meliloti which lacked NADP⁺ malic enzyme activity (dme) but retained NADP⁺ malic enzyme activity were isolated. In an otherwise wild-type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N₂. Structurally, these nodules appeared to develop like wild-type nodules up to the stage where N₂-fixation would normally begin. These results support the proposal that NAD⁺ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl-CoA required for TCA cycle function in N₂-fixing bacteroids which metabolize C₄-dicarboxylic acids supplied by the plant.     

17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae

Steroid metabolism studies have yielded evidence of 17β-hydroxysteroid dehydrogenase (17β-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17β-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17β-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17β-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E₂) and NADP⁺. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E₂ and NADP⁺. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP⁺ / NAD⁺ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17β-HSD and are consistent with the presence of more than one isoform of the enzyme.     

NAD-Linked Isocitrate Dehydrogenase: Isolation, Purification, and Characterization of the Protein from Pea Mitochondria

The NAD⁺-dependent isocitrate dehydrogenase from etiolated pea (Pisum sativum L.) mitochondria was purified more than 200-fold by dye-ligand binding on Matrix Gel Blue A and gel filtration on Superose 6. The enzyme was stabilized during purification by the inclusion of 20% glycerol. In crude matrix extracts, the enzyme activity eluted from Superose 6 with apparent molecular masses of 1400 ± 200, 690 ± 90, and 300 ± 50 kD. During subsequent purification steps the larger molecular mass species disappeared and an additional peak at 94 ± 16 kD was evident. The monomer for the enzyme was tentatively identified at 47 kD by sodium dodecyl-polyacrylamide gel electrophoresis. The NADP⁺-specific isocitrate dehydrogenase activity from mitochondria eluted from Superose 6 at 80 ± 10 kD. About half of the NAD⁺ and NADP⁺-specific enzymes remained bound to the mitochondrial membranes and was not removed by washing. The NAD⁺-dependent isocitrate dehydrogenase showed sigmodial kinetics in response to isocitrate (S_0.5 = 0.3 mm). When the enzyme was aged at 4°C or frozen, the isocitrate response showed less allosterism, but this was partially reversed by the addition of citrate to the reaction medium. The NAD⁺ isocitrate dehydrogenase showed standard Michaelis-Menten kinetics toward NAD⁺ (K_m = 0.2 mm). NADH was a competitive inhibitor (K_i = 0.2 mm) and, unexpectedly, NADPH was a noncompetitive inhibitor (K_i = 0.3 mm). The regulation by NADPH may provide a mechanism for coordination of pyridine nucleotide pools in the mitochondria.     

Determinants of Cofactor Specificity for the Glucose-6-Phosphate Dehydrogenase from Escherichia coli: Simulation,Kinetics and Evolutionary Studies

Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD⁺ and NADP⁺ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP⁺ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2''-phosphate of NADP⁺, but the same residues formed no observable interactions in the case of NAD⁺. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the k_cat/K_M value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP⁺, only R50 makes a contribution (about -1 kcal/mol) to NAD⁺ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP⁺ from NAD⁺. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP⁺-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD⁺ in the case of the NADP⁺-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.     

Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity

The cofactor-binding site of the NAD⁺-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2′- and 3′-hydroxyl groups of the adenosyl ribose ring of NAD⁺ and repels the 2′-phosphate moiety of NADP⁺. If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD⁺ is altered and rendered the enzyme capable of using NADP⁺. This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2′-phosphate of NADP⁺. Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP⁺. The double mutant ALDH3H1_{Glu149Thr/Ile200Val} showed a good catalysis with NADP⁺. Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a “closed” cofactor binding site. The cofactor specificity was shifted even further in favor of NADP⁺, as the mutant ALDH3H1_{E149T/V178R/I200V} uses NADP⁺ with almost 7-fold higher catalytic efficiency compared to NAD⁺. Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.     

Activity of NAD-dependent glucose-6-phosphate dehydrogenase in yeast-like organisms

Summary From tested yeast-like organisms, onlyGeotrichum candidum showed the same activity of glucose-6-phosphate dehydrogenase with both NAD⁺ and NADP⁺. i. e. 0.017–0.019 mol NADH/min. mg dry weight of cell free extracts. Omission of Mg⁺⁺ in the reaction mixture did not influence the activity of the enzyme in the presence of NAD⁺. Cell free extracts ofEndomyces magnusii showed only low activity of this enzyme and the ratio of its activity in the presence of NAD⁺ and NADP⁺, respectively, varied in individual cultures.Rhodotorula glutinis showed only an NADP⁺-dependent activity.     

Alteration of coenzyme specificity in halophilic NAD(P) glucose dehydrogenase by site-directed mutagenesis

Structural analysis of glucose dehydrogenase from Haloferax mediterranei revealed that the adenosine 2′-phosphate of NADP⁺ was stabilized by the side chains of Arg207 and Arg208. To investigate the structural determinants for coenzyme specificity, several mutants involving residues Gly206, Arg207 and Arg208 were engineered and kinetically characterized. The single mutants G206D and R207I were less efficient with NADP⁺ than the wild type, and the double and triple mutants G206D/R207I and G206D/R207I/R208N showed no activity with NADP⁺.In the single mutant G206D, the relation k_cat/K_NAD⁺ was 1.6 times higher than in the wild type, resulting in an enzyme that preferred NAD⁺ over NADP⁺. The single mutation was sufficient to modify coenzyme specificity, whereas other dehydrogenases usually required more than one or two mutations to change coenzyme specificity. However, the highest reaction rates were reached with the double mutant G206D/R207I and with coenzyme NAD⁺, where the k_cat was 1.6 times higher than the k_cat of the wild-type enzyme with NADP⁺. However, catalytic efficiency with NAD⁺ was lower, as the K_m value for coenzyme was 77 times higher than the wild type with NADP⁺.     

Glutamate Dehydrogenases: The Why and How of Coenzyme Specificity

NAD⁺ and NADP⁺, chemically similar and with almost identical standard oxidation–reduction potentials, nevertheless have distinct roles, NAD⁺ serving catabolism and ATP generation whereas NADPH is the biosynthetic reductant. Separating these roles requires strict specificity for one or the other coenzyme for most dehydrogenases. In many organisms this holds also for glutamate dehydrogenases (GDH), NAD⁺-dependent for glutamate oxidation, NADP⁺-dependent for fixing ammonia. In higher animals, however, GDH has dual specificity. It has been suggested that GDH in mitochondria reacts only with NADP(H), the NAD⁺ reaction being an in vitro artefact. However, contrary evidence suggests mitochondrial GDH not only reacts with NAD⁺ but maintains equilibrium using the same pool as accessed by β-hydroxybutyrate dehydrogenase. Another complication is the presence of an energy-linked dehydrogenase driving NADP⁺ reduction by NADH, maintaining the coenzyme pools at different oxidation–reduction potentials. Its coexistence with GDH makes possible a futile cycle, control of which is not yet properly explained. Structural studies show NAD⁺-dependent, NADP⁺-dependent and dual-specificity GDHs are closely related and a few site-directed mutations can reverse specificity. Specificity for NAD⁺ or for NADP⁺ has probably emerged repeatedly during evolution, using different structural solutions on different occasions. In various GDHs the P7 position in the coenzyme-binding domain plays a key role. However, whereas in other dehydrogenases an acidic P7 residue usually hydrogen bonds to the 2′- and 3′-hydroxyls, dictating NAD⁺ specificity, among GDHs, depending on detailed conformation of surrounding residues, an acidic P7 may permit binding of NAD⁺ only, NADP⁺ only, or in higher animals both.     

Glyceraldehyde-3-phosphate dehydrogenases from Euglena gracilis. Purification and physical and chemical characterization.

NAD⁺-dependent and NADP⁺-dependent glyceraldehyde-3-phosphate (G-3-P) dehydrogenases were isolated from Euglena gracilis and characterized as to their physical and chemical parameters. NAD⁺-G-3-P dehydrogenase was found to have a strong resemblance to similar enzymes from muscle tissue. It has a molecular weight of about 140,000, four subunits of identical size and charge, and a single species of NH₂-terminal amino acid. Two sulfhydryl groups per subunit are present, one of which is directly involved in the catalytic activity and is rapidly titratable. The enzyme also exhibits the “half the sites reactivity” of sulfhydryl groups as defined by O. P. Malhotra and S. A. Bernhard ((1968) J. Biol. Chem. 243, 1243). The pH and temperature optima are also similar to those of the enzymes from muscle tissue, as are the reaction kinetics and the strict specificity for NAD⁺.NADP⁺-dependent G-3-P dehydrogenase is different in many respects. Its molecular weight is slightly lower (~136,000) than that of the NAD⁺ enzyme, though it also consists of four subunits. It has a higher affinity for the reverse reaction substrates, in line with its probable function in vivo in CO₂ fixation. There is only one sulfhydryl group per subunit, and that is not involved in activity, suggesting a difference in reaction mechanisms between the two enzymes. The NADP⁺-dependent enzyme exhibits activation by ATP, whereas the NAD⁺-dependent enzyme is competitively inhibited by this nucleotide.The greatest difference observed is in the physical characteristics of the enzymes. NADP⁺-G-3-P dehydrogenase was highly hydrophobic. Its solubility in a 10% aqueous solution of p-dioxane was approximately four to five times that of the NAD⁺-enzyme. Isolation of the enzyme was accomplished by fractionation in 1,2-dimethoxyethane, which also stabilized the enzymatic activity, as did aqueous p-dioxane. The high axial ratio of the NADP⁺-enzyme (~9) coupled with its very low degree of hydration as well as the high degree of amidation of the dicarboxylic amino acids (>90%) indicates that the exterior of the enzyme molecule is probably hydrophobic in nature. This is in agreement with its in vivo hydrophobic environment in the chloroplast membrane and explains the lability of the enzyme once extracted into an aqueous environment as well as its stabilization in solvents.     

NADP+-specific 2-oxoglutarate dehydrogenase in denitrifying Pseudomonas species

Several denitrifying Pseudomonas strains contained an NADP⁺-specific 2-oxoglutarate dehydrogenase, in contrast to an NAD⁺-specific pyruvate dehydrogenase, if the cells were grown anaerobically with aromatic compounds. With non-aromatic substrates or after aerobic growth the coenzyme specificity of 2-oxoglutarate dehydrogenase changed to NAD⁺-specificity. The reaction stoichiometry and the apparent K _m-values of the enriched enzymes were determined: pyruvate 0.5 mM, coenzyme A 0.05 mM, NAD⁺ 0.25 mM; 2-oxoglutarate 0.6 mM, coenzyme A 0.05 mM, NADP⁺ 0.03 mM. Isocitrate dehydrogenase was NADP⁺-specific. The findings suggest that these strains contained at least two lipoamide dehydrogenases, one NAD⁺-specific, the other NADP⁺-specific.     

Studies on the metabolism of galactose-6-phosphate in rat heart and brain.

The subcellular distribution of NADP⁺ and NAD⁺-dependent glucose-6-phosphate and galactose-6-phosphate dehydrogenases were studied in rat liver, heart, brain, and chick brain. Only liver particulate fractions oxidized glucose-6-phosphate and galactose-6-phosphate with either NADP⁺ or NAD⁺ as cofactor. While all of the tissues examined had NADP⁺-dependent glucose-6-phosphate dehydrogenase activity, only rat liver and rat brain soluble fractions had NADP⁺-dependent galactose-6-phosphate dehydrogenase activity. Rat liver microsomal and rat brain soluble galactose-6-phosphate dehydrogenase activities were kinetically different (K_m's 0.5 mm and 10 mm, respectively, for galactose-6-phosphate), although their reaction products were both 6-phosphogalactonate. Rat brain subcellular fractions did not oxidize 6-phosphogalactonate with either NADP⁺ or NAD⁺ cofactors but phosphatase activities hydrolyzing 6-phosphogalactonate, galactose-6-phosphate and galactose-1-phosphate were found in crude brain homogenates. In addition, galactose-6-phosphate and 6-phosphogalactonate were tested as inhibitors of various enzymes, with largely negative results, except that 6-phosphogalactonate was a competitive inhibitor (K_i = 0.5 mM) of rat brain 6-phosphogluconate dehydrogenase.     

A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71

In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD⁺-specific (NAD-IDH) or NADP⁺-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD⁺-specific and showed no detectable activity with NADP⁺. The K _m values of the enzyme for NAD⁺ were 262.6±7.4 μM or 309.1±11.2 μM with Mg²⁺ or Mn²⁺ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP⁺ was approximately 24-fold higher than that for NAD⁺, suggesting that ClIDH is an NAD⁺-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn²⁺. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD⁺-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs.     

Purification and kinetic characterization of γ-aminobutyraldehyde dehydrogenase from rat liver

Oxidative deamination of putrescine, the precursor of polyamines, gives rise to γ-aminobutyraldehyde (ABAL). In this study an aldehyde dehydrogenase, active on ABAL, has been purified to electrophoretic homogeneity from rat liver cytoplasm and its kinetic behaviour investigated. The enzyme is a dimer with a subunit molecular weight of 51,000. It is NAD⁺-dependent, active only in the presence of sulphhydryl compounds and has a pH optimum in the range 7.3–8.4. Temperatures higher than 28°C promote slow activation and the process is favoured by the presence of at least one substrate. K_m for aliphatic aldehydes decreases from 110 μM for ABAL and acetaldehyde to 2–3 μM for capronaldehyde. The highest relative V-values have been observed with ABAL (100) and isobutyraldehyde (64), and the lowest with acetaldehyde (14). Affinity for NAD⁺ is affected by the aldehyde present at the active site: K_m for NAD⁺ is 70 μM with ABAL, 200 μM with isobutyraldehyde and capronaldehyde, and>800 μM with acetaldehyde. The kinetic behaviour at 37°C is quite complex; according to enzymatic models, NAD⁺ activates the enzyme (K_act 500 μM) while NADH competes for the regulatory site (K_in 70 μM). In the presence of high NAD⁺ concentrations (4 mM), ABAL promotes further activation by binding to a low-affinity regulatory site (K_act 10 mM). The data show that the enzyme is probably an E₃ aldehyde dehydrogenase, and suggest that it can effectively metabolize aldehydes arising from biogenic amines.     

NMR solution structures of Runella slithyformis RNA 2′-phosphotransferase Tpt1 provide insights into NAD+ binding and specificity

Tpt1, an essential component of the fungal and plant tRNA splicing machinery, catalyzes transfer of an internal RNA 2′-PO₄ to NAD⁺ yielding RNA 2′-OH and ADP-ribose-1′,2′-cyclic phosphate products. Here, we report NMR structures of the Tpt1 ortholog from the bacterium Runella slithyformis (RslTpt1), as apoenzyme and bound to NAD⁺. RslTpt1 consists of N- and C-terminal lobes with substantial inter-lobe dynamics in the free and NAD⁺-bound states. ITC measurements of RslTpt1 binding to NAD⁺ (K_D ∼31 μM), ADP-ribose (∼96 μM) and ADP (∼123 μM) indicate that substrate affinity is determined primarily by the ADP moiety; no binding of NMN or nicotinamide is observed by ITC. NAD⁺-induced chemical shift perturbations (CSPs) localize exclusively to the RslTpt1 C-lobe. NADP⁺, which contains an adenylate 2′-PO₄ (mimicking the substrate RNA 2′-PO₄), binds with lower affinity (K_D ∼1 mM) and elicits only N-lobe CSPs. The RslTpt1·NAD⁺ binary complex reveals C-lobe contacts to adenosine ribose hydroxyls (His99, Thr101), the adenine nucleobase (Asn105, Asp112, Gly113, Met117) and the nicotinamide riboside (Ser125, Gln126, Asn163, Val165), several of which are essential for RslTpt1 activity in vivo. Proximity of the NAD⁺ β-phosphate to ribose-C1″ suggests that it may stabilize an oxocarbenium transition-state during the first step of the Tpt1-catalyzed reaction.     

Analysis of glycerol dehydrogenase activities present in <Emphasis Type="Italic">Mucor circinelloides</Emphasis> YR-1

Two inducible NADP⁺-dependent glycerol dehydrogenase (GlcDH) activities were identified in Mucor circinelloides strain YR-1. One of these, denoted iGlcDH2, was specifically induced by n-decanol when it was used as sole carbon source in the culture medium, and the second, denoted iGlcDH1, was induced by alcohols and aliphatic or aromatic hydrocarbons when glycerol was used as the only substrate. iGlcDH2 was found to have a much broader substrate specificity than iGlcDH1, with a low activity as an ethanol dehydrogenase with NAD⁺ or NADP⁺ as cofactor. Both isozymes showed an optimum pH for activity of 9.0 in Tris-HCl buffer and are subject to carbon catabolite repression. In contrast, the constitutive NADP⁺-dependent glycerol dehydrogenases (GlcDHI, II, and III) were only present in cell extracts when the fungus was grown in glycolytic carbon sources or glycerol under oxygenation, and their optimum pH was 7.0 in Tris-HCl buffer. In addition to these five NADP⁺-dependent glycerol dehydrogenases, a NAD⁺-dependent alcohol dehydrogenase is also present in glycerol or n-decanol medium; this enzyme was found to have weak activity as a glycerol dehydrogenase.