Threonine 188 is critical for interaction with NAD+ in human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins. It is a member of the short-chain dehydrogenase family of enzymes. A relatively conserved threonine residue corresponding to threonine 188 of 15-PGDH is proposed to be involved in the interaction with the carboxamide group of NAD+. Site-directed mutagenesis was used to examine the important role of this residue. Threonine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that of the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE2 was similar to the wild enzyme; however, the Km value for NAD+ was increased over 100 fold. These results suggest that threonine 188 is critical for interaction with NAD+ and contributes to the full catalytic activity of 15-PGDH.     

Histidine 21 is at the NAD+ binding site of diphtheria toxin

Treatment of fragment A chain of diphtheria toxin (DT-A) with diethylpyrocarbonate modifies His-21, the single histidine residue present in the chain, without alteration of other residues. Parallel to histidine modification, NAD+ binding and the NAD-glycohydrolase and ADP-ribosyltransferase activities of DT-A are lost. Both NAD+ and adenosine are very effective in protecting DT-A from histidine modification and in preserving its biological properties, while adenine is ineffective. Reversal of histidine modification with hydroxylamine restores both NAD+ binding and enzymatic activities of the toxin. The possible role of His-21 in the activity of diphtheria toxin is discussed in relation to the available three-dimensional structure of the related toxin produced by Pseudomonas aeruginosa.     

Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site-directed mutagenesis.

The 2',3'-dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD+ binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (Kd = 0.46 mM for the fast phase, 0.45 mM for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min-1 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme-oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (> 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme-oADP complex was previously reduced by NaBH4, suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD+, NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [14C]oADP per mol of subunit was incorporated. Cleavage of [14C]oADP-modified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the MODELLER 4 program. The model confirmed that Lys360 is positioned at the NAD+-binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys360-->Ala enzyme exhibited unchanged kcat and Km values for formate but showed reduced affinity for NAD+. The molecular model was used to help interpret these biochemical data concerning the Lys360-->Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.     

Cysteinyl peptide labeled by 3-bromo-2-ketoglutarate in the active site of pig heart NAD+-dependent isocitrate dehydrogenase

The substrate affinity label 3-bromo-2-ketoglutarate (BrKG) reacts covalently with pig heart NAD+-specific isocitrate dehydrogenase with complete inactivation and incorporation of about 0.8 mol of reagent/mol of average enzyme subunit [Bednar, R.A., Hartman, F.C., & Colman, R.F. (1982) Biochemistry 21, 3681-3689]. Protection against inactivation is provided by isocitrate and Mn2+. We have now identified a critical modified peptide by comparison of the peptides labeled by BrKG at pH 6.1 in the absence and presence of isocitrate and Mn2+. Modified enzyme, isolated from unreacted BrKG, was incubated with [3H]NaBH4 to reduce the keto group of protein-bound 2-ketoglutarate and thereby introduce a radioactive tracer into the modified amino acid. Following carboxymethylation and digestion with trypsin, the specific modified peptide was isolated by reverse-phase HPLC, first in 0.1% trifluoroacetic acid with a gradient in acetonitrile and then in 20 mM ammonium acetate, pH 5.8, with an acetonitrile gradient. Gas-phase sequencing gave the modified peptide: Ser-Ala-X-Val-Pro-Val-Asp-Phe-Glu-Glu-Val-Val-Val-Ser-Ser-Asn-Ala-Asp-Gl u-Glu- Asp-Ile-Arg. The corresponding tryptic peptide that was isolated from unmodified enzyme yielded the same sequence except for (carboxymethyl)cysteine at position 3, suggesting that cysteine is the target of 3-bromo-2-ketoglutarate. Pig heart NAD+-dependent isocitrate dehydrogenase is composed of three distinct subunits (alpha, beta, and gamma) that can be separated by chromatofocusing in urea and identified by analytical gel isoelectric focusing. The peptide modified by 3-bromo-2-ketoglutarate, which is in or near the substrate site, is derived only from the separated gamma subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subunit interactions of yeast NAD+-specific isocitrate dehydrogenase

Yeast mitochondrial NAD(+)-specific isocitrate dehydrogenase is an octamer composed of four each of two nonidentical but related subunits designated IDH1 and IDH2. IDH2 was previously shown to contain the catalytic site, whereas IDH1 contributes regulatory properties including cooperativity with respect to isocitrate and allosteric activation by AMP. In this study, interactions between IDH1 and IDH2 were detected using the yeast two-hybrid system, but interactions between identical subunit polypeptides were not detected with this or other methods. A model for heterodimeric interactions between the subunits is therefore proposed for this enzyme. A corollary of this model, based on the three-dimensional structure of the homologous enzyme from Escherichia coli, is that some interactions between subunits occur at isocitrate binding sites. Based on this model, two residues (Lys-183 and Asp-217) in the regulatory IDH1 subunit were predicted to be important in the catalytic site of IDH2. We found that individually replacing these residues with alanine results in mutant enzymes that exhibit a drastic reduction in catalysis both in vitro and in vivo. Also based on this model, the two analogous residues (Lys-189 and Asp-222) of the catalytic IDH2 subunit were predicted to contribute to the regulatory site of IDH1. A K189A substitution in IDH2 was found to produce a decrease in activation of the enzyme by AMP and a loss of cooperativity with respect to isocitrate. A D222A substitution in IDH2 produces similar regulatory defects and a substantial reduction in V(max) in the absence of AMP. Collectively, these results suggest that the basic structural/functional unit of yeast isocitrate dehydrogenase is a heterodimer of IDH1 and IDH2 subunits and that each subunit contributes to the isocitrate binding site of the other.     

Kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase

The kinetic mechanism of Halobacterium halobium NAD+-glutamate dehydrogenase (EC 1.4.1.3) has been investigated at pH 9.0, 3 M NaCl and 40 degrees C in both directions, by initial rate and inhibition studies. The results of the initial rate studies indicate that the mechanism is sequential with respect to substrate addition. The inhibition patterns obtained with halophilic NAD+-glutamate dehydrogenase are not consistent with a simple ordered mechanism without modification. They can, however, be reconciled with this type of mechanism by postulating an appropriate abortive complex.     

A disubstituted NAD+ analogue is a nanomolar inhibitor of trypanosomal glyceraldehyde-3-phosphate dehydrogenase

N6-Naphthalenemethyl-2'-methoxybenzamido-beta-NAD+, a derivative of a low micromolar first-generation inhibitor of trypanosomal glyceraldehyde phosphate dehydrogenase (GAPDH), was synthesized, taking advantage of methodology for the selective phosphitylation of nucleosides. The compound was found to be a poor alternate cosubstrate for GAPDH, but an extremely potent inhibitor. Although intended for use in crystallization trials, the analogue presents possibilities for further drug design.     

Conformation of NAD+ bound to allosteric L-lactate dehydrogenase activated by chemical modification

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilus L-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+ bound to the modified enzyme in the absence of Fru-1,6-P2 was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+ were in the anti conformation, the ribose rings being in the C3'-endo form. This conformation was almost the same as that of NAD+ bound to the native enzyme-Fru-1,6-P2 complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3'-endo-anti form of the enzyme-bound NAD+ is essential for the activation of the T. caldophilus L-lactate dehydrogenase.     

A new NAD+-dependent glyceraldehyde dehydrogenase obtained by rational design of l-lactaldehyde dehydrogenase from Escherichia coli

NAD + -dependent glyceraldehyde dehydrogenases usually had lower activity in the nonphosphorylated Entner–Doudoroff (nED) pathway. In the present study, a new NAD + -dependent glyceraldehyde dehydrogenase was engineered from l-lactaldehyde dehydrogenase of E. coli (EC: 1.2.1.22). Through comparison of the sequence alignment and the active center model, we found that a residue N286 of l-lactaldehyde dehydrogenase contributed an important structure role to substrate identification. By free energy calculation, three mutations (N286E, N286H, N286T) were chosen to investigate the change of substrate specificity of the enzyme. All mutants were able to oxidate glyceraldehyde. Especially, N286T showed the highest activity of 1.1U/mg, which was 5-fold higher than the reported NAD + -dependent glyceraldehyde dehydrogenases, and 70% activity was retained at 55?°C after an hour. Compared to l-lactaldehyde, N286T had a one-third lower K_m value to glyceraldehyde.     

Structural map of the dicyclohexylcarbodiimide site of chloroplast coupling factor determined by resonance energy transfer

Fluorescence resonance energy-transfer measurements were made on the membrane-bound chloroplast coupling factor. The distances from the N,N'-dicyclohexylcarbodiimide-binding site on the membrane-bound portion of the enzyme (CF0) to the vesicle surface and to two sulfhydryl sites on the gamma-polypeptide were determined. The dicyclohexylcarbodiimide-binding site was labeled with the fluorescent species N-cyclohexyl-N'-pyrenylcarbodiimide. The vesicle surface was labeled with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine. Steady-state energy transfer between the fluorescent-labeled enzyme (energy donor) and varying concentrations of the ethanolamine derivative (energy acceptor) indicated that the distance of closest approach between the energy donor and the outer vesicle surface is 16-24 A. Two specific sites on the gamma-polypeptide were reacted with a coumarinylmaleimide derivative; one is a sulfhydryl that can be labeled only on the thylakoids under energized conditions (the "light" site), while the other is the disulfide site that regulates enzymatic activity. Energy-transfer measurements utilizing steady-state fluorescence and fluorescence lifetime methods indicated that the dicyclohexylcarbodiimide site is approximately 41 A from the light site and approximately 50 A from the gamma-disulfide site. These distances are used to extend the current structural model of the chloroplast coupling factor.     

Spectrofluorometric assay of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase activity

A simple, rapid, and sensitive spectrofluorometric assay for 15-hydroxyprostaglandin dehydrogenase activity was developed in which the rate of production of NADH was monitored. The cytosolic fraction prepared from human placental tissue was employed as the enzyme source. The assay was conducted at pH 9.5 since 15-ketoprostaglandin Δ¹³-reductase and NADH oxidase activities were inhibited at this pH, thereby minimizing the interference of the reactions catalyzed by these enzymes in the assay of prostaglandin dehydrogenase activity.     

Isotope effects on binding of NAD+ to lactate dehydrogenase

The isotope effect on binding [4-2H]NAD+ and [4-3H]NAD+ to lactate dehydrogenase has been shown to be 1.10 +/- 0.03 by whole molecule isotope ratio mass spectrometry and 1.085 +/- 0.01 by 3H/14C scintillation counting. These values demonstrate that specific interactions of the nicotinamide ring with the enzyme make the C-H bond at C-4 less stiff in the binary complex.     

Improving the purification of NAD+-dependent formate dehydrogenase from Candida methylica

The Candida methylica (cm) recombinant wild type formate dehydrogenase (FDH) gene has been cloned into the pQE-2 TAGZyme expression vector and the 6xHis-tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N-terminal His-tag. After the purification procedure, 1.2 mg/mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.     

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.