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PubMed1997

Structural and catalytic properties of the expressed and purified NAD(H)- and NADP(H)-binding domains of proton-pumping transhydrogenase from Escherichia coli.

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains three domains: the hydrophilic domains I and III harbor the binding sites for NAD(H) and NADP(H), respectively, and domain II represents the membrane-spanning region. Proton translocation involves primarily domain II but possibly also domain III, which contains the essential betaAsp392 residue. In the present investigation, the major portions of domain I (EcTHSalpha1 and EcTHSalpha2) and domain III (EcTHSbeta1) were overexpressed in E. coli and purified therefrom. EcTHSbeta1 was purified mainly in its holoform containing approximately 95% NADP+ and 5% NADPH. When combined, EcTHSalpha1/EcTHSalpha2 and EcTHSbeta1 were catalytically active, indicating native-like structures. Due to the lack of structural information and its possible role in proton pumping, EcTHSbeta1 was primarily characterized. Substrate-binding characteristics and conformational changes were investigated by fluorescence and CD. Fluorescence arising from the single betaTrp415 of EcTHSbeta1 was quenched upon binding of NADPH by resonance energy transfer, an effect that provides an important tool for investigating substrate interactions with this domain and the determination of Kd values. The apparent relative binding affinity for NADPH was found to be about 50 times higher than that for NADP+. Circular dichroism was used to estimate secondary structure content and for conformational analysis of EcTHSbeta1 in the absence and presence of added substrates at various temperatures. Results show that domain III complexed with NADPH or NADP+ adopts different conformations. Isoelectric focusing and native gel electrophoresis experiments support this finding. It is proposed that these structural differences play a central role in a conformationally-driven proton pump mechanism of the intact enzyme.

Amino Acid Sequence↗
PubMed2002

Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I-domain III interactions in proton-translocating Escherichia coli transhydrogenase.

Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction). This reaction is stimulated by an electrochemical proton gradient, Delta p, presumably through an increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively. Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III. With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues beta A398, beta S404, beta I406, beta G408, beta M409 and beta V411 in loop D, and residue beta Y431 in loop E were selected. In addition, the previously made mutants betaD392C and betaT393C in loop D, and beta G430C and beta A432C in loop E, were included. All loop D and E mutants, especially beta I406C and beta G430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild-type enzyme. Determination of values indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E. In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates. Most domain III mutants also showed a decreased affinity for domain I. The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III.

Catalysis↗
PubMed1998

Probing the function of the invariant glutamyl residue 312 in spinach ferredoxin-NADP+ reductase.

Ferredoxin-NADP+ reductase, the prototype of a large family of structurally related flavoenzymes, pairs single electrons carried by ferredoxin I and transfers them as a hydride to NADP+. Four mutants of the enzyme, in which Glu-312 was replaced with Asp, Gln, Leu, and Ala to probe the role of the residue charge, size, and polarity in the enzyme activity, have been heterologously expressed, purified, and characterized through steady-state, rapid kinetic studies, ligand-binding experiments, and three-dimensional structure determination by x-ray crystallography. The E312L mutant was the only one that was almost inactive (approximately 1%), whereas unexpectedly the E312A reductase was 10-100% active with the various acceptors tested. Rapid kinetic absorption spectroscopy studies demonstrated that flavin reduction by NADPH was impaired in the mutants. Furthermore, NADP(H) binding was partially perturbed. These functional and structural studies lead us to conclude that Glu-312 does not fulfil the role of proton donor during catalysis, but it is required for proper binding of the nicotinamide ring of NADP(H). In addition, its charge modulates the two one-electron redox potentials of the flavin to stabilize the semiquinone form.

Amino Acid Sequence↗
PubMed1991

Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family.

The three-dimensional structure of spinach ferredoxin-NADP+ reductase (NADP+, nicotinamide adenine dinucleotide phosphate) has been determined by x-ray diffraction at 2.6 angstroms (A) resolution and initially refined to an R factor of 0.226 at 2.2 A resolution. The model includes the flavin-adenine dinucleotide (FAD) prosthetic group and the protein chain from residue 19 through the carboxyl terminus at residue 314 and is composed of two domains. The FAD binding domain (residues 19 to 161) has an antiparallel beta barrel core and a single alpha helix for binding the pyrophosphate of FAD. The NADP binding domain (residues 162 to 314) has a central five-strand parallel beta sheet and six surrounding helices. Binding of the competitive inhibitor 2'-phospho-AMP (AMP, adenosine monophosphate) places the NADP binding site at the carboxyl-terminal edge of the sheet in a manner similar to the nucleotide binding of the dehydrogenase family. The structures reveal the key residues that function in cofactor binding and the catalytic center. With these key residues as a guide, conclusive evidence is presented that the ferredoxin reductase structure is a prototype for the nicotinamide dinucleotide and FAD binding domains of the enzymes NADPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADH-cytochrome b5 reductase, and NADH-nitrate reductase. Thus this structure provides a structural framework for the NADH- or NADPH-dependent flavoenzyme parts of five distinct enzymes involved in photosynthesis, in the assimilation of inorganic nitrogen and sulfur, in fatty-acid oxidation, in the reduction of methemoglobin, and in the metabolism of many pesticides, drugs, and carcinogens.

Adenosine Diphosphate↗
PubMed1980

Energy-linked transhydrogenation from NADPH to [14C]NADP.

Submitochondrial particles catalyze transhydrogenation from NADPH to [14C]NADP. This transhydrogenation is energy-linked, since its rate increases several-fold when the system is energized by succinate oxidation in the presence of rotenone (inhibitable by antimycin A or uncouplers), or by ATP hydrolysis (inhibitable by rutamycin or uncouplers). As in the case of transhydrogenation reactions from NAD(P)H to 3-ace-tylpyridine adenine dinucleotide phosphate and to thionicotinamide adenine dinucleotide phosphate, transhydrogenation from NADPH to [14C]NADP is also sensitive to treatment of the particles with trypsin or the arginyl residue modifier, butanedione. However, unlike the former reactions, transhydrogenation from NADPH to [14C]NADP cannot accumulate energy in the concentrations of the products, because, except for radioactivity, the nature and concentrations of the reactants and products remain unchanged throughout the course of the reaction. Therefore, the unrecoverable energy utilization by this region could be ascribed to an entropic component of the process, very likely an enzyme conformation change necessary for facilitation of hydride ion transfer from NADPH to [14C]NADP. This interpretation is in agreement with our previous kinetic evidence for enzyme conformation change associated with energy-linked transhydrogenation from NADH to 3-acetylpyridine adenine dinucleotide phosphate and thionicotinamide adenine dinucleotide phosphate, and with our conclusions regarding the mechanism of action of the transhydrogenase enzyme (Galante, Y.M., Lee, Y., and Hatefi, Y. (1980) J. Biol. Chem. 255, 9641-9646).

Animals↗
PubMed1980

Energy-linked mitochondrial transhydrogenation from NADPH to NADP analogs.

The mitochondrial energy-linked transhydrogenase enzyme catalyzes hydride ion transfer between NAD and HADP, of which the reaction NADH leads to NADP is slow in the absence of energy and is accelerated 10-fold or more when the mitochondrial membrane is energized by ATP hydrolysis or respiration. The enzyme is a proton pump and effects proton translocation coupled to hydride ion transfer from NADPH to NAD (Earle, S.R., and Fisher, R.R. (1980) J. Biol Chem. 255, 827-830). The present studies have shown that submitochondrial particles also catalyze transhydrogenation from NADPH to two NADP analogs, namely 3-acetylpyridine adenine dinucleotide phosphate (AcPyADP) and thionicotinamide adenine dinucleotide phosphate (thioNADP). Both reaction rates are greatly accelerated when the system is energized by ATP hydrolysis (inhibitable by uncouplers or rutamycin) or succinate oxidation (inhibitable by uncouplers or antimycin A). As in the case of NAD(H) in equilibrium with NADP(H) reactions, the transhydrogenations from NADPH to AcPyADP and thioNADP are inhibited by treatment of submitochondrial particles with trypsin or the arginyl residue modifier, butanedione. The Km values of the above substrates and the Vmax values under energy-linked conditions have been determined. The finding that the mitochondrial energy-linked transhydrogenase enzyme catalyzes transhydrogenation from NADPH to NADP analogs has revealed features regarding substrate site specificities and the effect of substrates on the directionality of proton translocation by the enzyme.

Animals↗
PubMed1994

Identification and characterization of the second regulatory disulfide bridge of recombinant sorghum leaf NADP-malate dehydrogenase.

Unique among malate dehydrogenases, the NADP-dependent chloroplastic form undergoes a reductive activation in the light. This process is thioredoxin-mediated and involves at least two disulfides. Only one of them, situated near the N terminus, has been localized. The enzyme also bears 2 cysteines at the C terminus. The possible role of these cysteines was investigated by replacing them separately, or together, by alanines, by site-directed mutagenesis. The proteins altered at the C terminus were still dithiol-dependent for full activation, with activation kinetics similar to those of the wild type enzyme. However, they exhibited a weak activity in the oxidized form with a dramatically increased Km for oxalacetate. Their activation was not inhibited by NADP. When C-terminal Cys mutations were combined with N-terminal Cys mutations, permanently active, thioredoxin-independent enzymes were obtained. They exhibited the biochemical properties of the activated wild type protein. Clearly, the 2 C-terminal cysteines constitute the second thioredoxin-dependent regulatory disulfide of NADP-malate dehydrogenase. Integrating our data about the characteristics of each of the regulatory disulfides and information from three-dimensional structure modeling, we propose a model for the redox control of NADP-malate dehydrogenase.

Amino Acid Sequence↗
PubMed1979

Alcohol dehydrogenase (ADH) in yeasts. II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica.

In Sm. lipolytica one NAD+-dependent and three NADP+-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD+-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD+-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP+-dependent alcohol dehydrogenases (ADH II--IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP+-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP+-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allylalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.

Acetates↗
PubMed1994

Further characterization by site-directed mutagenesis of the protein-protein interface in the ferredoxin/ferredoxin:NADP+ reductase system from Anabaena: requirement of a negative charge at position 94 in ferredoxin for rapid electron transfer.

Ferredoxins are small electron transfer proteins found ubiquitously in nature. In green plant photosynthesis, the soluble [2Fe-2S] ferredoxin shuttles electrons from Photosystem I to ferredoxin:NADP+ reductase. In order to define the features of the protein/protein interface required for efficient electron transfer from ferredoxin to ferredoxin:NADP+ reductase, we have made site-directed mutants of the ferredoxin from the cyanobacterium Anabaena 7120 and measured the rate constants for electron transfer to ferredoxin:NADP+ reductase using laser flash photolysis. Previous results from this laboratory identified two residues in ferredoxin that were crucial to electron transfer between these proteins. One such position (F65) was subsequently shown to require an aromatic amino acid, and it was further shown that interprotein electron transfer was rate-limiting in the case of the slowly reacting F65A mutant (Hurley et al., 1993 J. Am. Chem. Soc. 115, 11,698-11,701). The second crucial position (E94) is shown in the present study to require a negative charge in order to maintain wild-type-like electron transfer reactivity toward ferredoxin:NADP+ reductase. Further, we also demonstrate, for the slowly reacting E94Q mutant, that electron transfer is the rate-limiting step in the interprotein interaction.

Anabaena↗
PubMed1995

N-ethylmaleimide profiling of yeast NADP-dependent isocitrate dehydrogenase.

Yeast NADP-dependent isocitrate dehydrogenase is inactivated by N-ethyl-maleimide (NEM) at pH 7.7 and 30 degrees C. Reaction with cysteine382 occurs most rapidly and is accompanied by loss of about 50% of the enzymatic activity. A slower phase of inactivation ensues during which lysine343 is the major target of NEM, while minor products result from reaction at cysteine73 and cysteine354. Protection against the second phase of inactivation is provided by NADP, NAPH, or manganous-isocitrate. Comparison of the time-dependence of inactivation and the products of reaction with N-ethyl-ethylmaleimide (NEM profiling) of the pig heart (G. E. Smyth and R. F. Colman, 1991, J. Biol. Chem. 266, 14918-14925) and yeast NADP-specific isocitrate dehydrogenases have been coupled with an examination of the crystal structure of the Escherichia coli isocitrate dehydrogenase. The following conclusions have been reached: while no cysteine is essential for activity, yeast Cys382/pig Cys379 is close to the adenine portion of the NADP binding site, and pig Cys269 is located in the region of the metal-isocitrate binding site.

Amino Acid Sequence↗
PubMed1995

NAD+/NADP+-dependent malic enzyme: evidence of a NADP+ preferring activity in human skeletal muscle.

The L-malate NAD(P)+ oxidoreductase (decarboxylating) E.C.1.1.1.39 was purified from human skeletal muscle; the specific activity estimated in the presence of NAPD+ as coenzyme was approximately 15 mumol/min/mg. The apparent Vmax values for NAD+ (approximately 8 mumol/min/mg) and NADP+ (approximately 16 mumol/min/mg) show that the enzyme (in this tissue) is more active in the presence of NAPD+. This observation was confirmed by the estimation of enzymatic activity in competition experiments where both NAD+ and NADP+ were used together as coenzymes. The absence of pyruvate carboxylation and of oxalacetate decarboxylation activities demonstrates that the enzyme studied is E.C.1.1.1.39. In addition, the apparent Km values for NAD+ and NADP+ were calculated (15 and 0.05 mM, respectively). This paper provides the first demonstration of a NADP+ preferring activity of the enzyme in human skeletal muscle.

Carboxy-Lyases↗
PubMed2000

The crystal structure of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaeon Methanothermus fervidus in the presence of NADP(+) at 2.1 A resolution.

The crystal structure of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the archaeon Methanothermus fervidus has been solved in the holo form at 2.1 A resolution by molecular replacement. Unlike bacterial and eukaryotic homologous enzymes which are strictly NAD(+)-dependent, GAPDH from this organism exhibits a dual-cofactor specificity, with a marked preference for NADP(+) over NAD(+). The present structure is the first archaeal GAPDH crystallized with NADP(+). GAPDH from M. fervidus adopts a homotetrameric quaternary structure which is topologically similar to that observed for its bacterial and eukaryotic counterparts. Within the cofactor-binding site, the positively charged side-chain of Lys33 decisively contributes to NADP(+) recognition through a tight electrostatic interaction with the adenosine 2'-phosphate group. Like other GAPDHs, GAPDH from archaeal sources binds the nicotinamide moiety of NADP(+) in a syn conformation with respect to the adjacent ribose and so belongs to the B-stereospecific class of oxidoreductases. Stabilization of the syn conformation is principally achieved through hydrogen bonding of the carboxamide group with the side-chain of Asp171, a structural feature clearly different from what is observed in all presently known GAPDHs from bacteria and eukaryotes. Within the catalytic site, the reported crystal structure definitively confirms the essential role previously assigned to Cys140 by site-directed mutagenesis studies. In conjunction with new mutation results reported in this paper, inspection of the crystal structure gives reliable evidence for the direct implication of the side-chain of His219 in the catalytic mechanism. M. fervidus grows optimally at 84 degrees C with a maximal growth temperature of 97 degrees C. The paper includes a detailed comparison of the present structure with four other homologous enzymes extracted from mesophilic as well as thermophilic organisms. Among the various phenomena related to protein thermostabilization, reinforcement of electrostatic and hydrophobic interactions as well as a more efficient molecular packing appear to be essentially promoted by the occurrence of two additional alpha-helices in the archaeal GAPDHs. The first one, named alpha4, is located in the catalytic domain and participates in the enzyme architecture at the quaternary structural level. The second one, named alphaJ, occurs at the C terminus and contributes to the molecular packing within each monomer by filling a peripherical pocket in the tetrameric assembly.

Amino Acid Sequence↗
PubMed1993

One-step purification of plant ferredoxin-NADP+ oxidoreductase expressed in Escherichia coli as fusion with glutathione S-transferase.

Complementary DNA sequences encoding the mature form of pea ferredoxin-NADP+ reductase were cloned in-frame at the 3' end of the Schistosoma japonicum glutathione S-transferase gene in the expression vector pGEX-3X (Smith and Johnson, Gene 67, 31-40, 1988). A spacer sequence linking the two genes was modified to provide a proteolytic site just before the first amino acid residue of mature pea reductase. When introduced into competent Escherichia coli cells and induced, the resulting plasmid (pGF205) directed the expression of a 60-kDa immunoreactive peptide that results from the fusion between glutathione S-transferase and ferredoxin-NADP+ reductase sequences. The fused protein could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione. It showed both transferase and reductase activities. Removal of the transferase portion by cleavage with the restriction protease Xa rendered ferredoxin-NADP+ reductase electrophoretically homogeneous. The purified transgenic enzyme showed kinetic and spectroscopic properties that were similar to those reported for the plant flavoprotein, indicating that, even when fused to the 27-kDa transferase portion, the reductase was still able to assemble FAD and to acquire an active conformation in the bacterial host. The expression-purification protocol employed here allows the isolation of up to 1 mg of active ferredoxin-NADP+ reductase/g of transformed cells. The system is potentially useful for the purification of activity-impaired forms of the flavoprotein.

Amino Acid Sequence↗
PubMed1995

The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene.

The gdhA gene of Synechocystis PCC 6803, which encodes an NADP-dependent glutamate dehydrogenase (NADP-GDH), has been cloned by complementation of an Escherichia coli glutamate auxotroph. This gene was found to code for a polypeptide of 428 amino acid residues, whose sequence shows high identity with those of archaebacteria (42-47%), some Gram-positive bacteria (40-44%) and mammals (37%). The minimal fragment of Synechocystis DNA required for complementation (2kb) carries the gdhA gene preceded by an open reading frame (ORF2) encoding a polypeptide of 130 amino acids. ORF2 and gdhA are co-transcribed as a 1.9 kb mRNA, but shorter transcripts including only gdhA were also detected. Two promoter regions were identified upon transcriptional fusion to the cat reporter gene of a promoter probe plasmid. Transcription from the promoter upstream of ORF2 was found to be regulated depending on the growth phase of Synechocystis, in parallel to NADP-GDH activity. This promoter is expressed in Escherichia coli too, in contrast to the second promoter, located between ORF2 and gdhA, which was silent in E. coli and did not respond to the stage of growth in Synechocystis. Disruption of the cyanobacterial gdhA gene with a chloramphenicol resistance cassette yielded a mutant strain totally lacking NADP-GDH activity, demonstrating that this gene is not essential to Synechocystis 6803 under our laboratory conditions.

Amino Acid Sequence↗
PubMed1980

Photokinetic microanalysis of NADP+, using bacterial luciferase.

Bioluminescence photokinetic assay of NADP+ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. the analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP+ as measurable product and for nucleotide assay in cell samples of 0.5--10 microgram dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP+. Previously, FMN, NADH, NAD+ and NADH have been analysed with the bacterial luciferase system. Its applicability has not been extended by the assay of NADP+.

Enzymes↗
PubMed1986

Interaction of ferredoxin with ferredoxin:NADP reductase: effects of chemical modification of ferredoxin.

Chemical modification studies have been conducted on spinach ferredoxin to determine the nature of the groups on ferredoxin involved in its interaction with its reaction partners. Modification of a limited number (three or four) carboxyl groups or of the single histidine residue resulted in a decreased ability of ferredoxin to participate in NADP photoreduction but not in cytochrome c photoreduction, suggesting that these groups may be involved in interaction with ferredoxin:NADP reductase but are not involved in interaction with the reducing side of Photosystem I. In contrast, modification of amino groups or the single arginine residue on ferredoxin had little effect on the ability of ferredoxin to participate in NADP photoreduction, suggesting these groups are not involved in the interaction of ferredoxin with either ferredoxin:NADP reductase or the reducing side of Photosystem I. Attempts to modify tyrosine residues on ferredoxin resulted in destruction of the iron-sulfur center of the protein.

Arginine↗
PubMed1990

Rhythmic changes in the activities of NAD kinase and NADP phosphatase in the achlorophyllous ZC mutant of Euglena gracilis Klebs (strain Z).

NAD kinase and NADP phosphatase activities were detected in the supernatant and the pellet fractions prepared by sonication and centrifugation of the achlorophyllous ZC mutant of the phytoflagellate Euglena gracilis. A detailed study of substrate concentration-velocity curves enabled us to define the saturating substrate concentrations that were used in the enzyme assays. An analysis of the reproducibility of the entire assay procedure indicated that the pooled standard error was about 14%. We report circadian variations in the activities of NAD kinase and NADP phosphatase in the soluble and membrane-bound fractions of both synchronously dividing and nondividing cultures maintained in constant darkness. Bimodal circadian rhythms in total NADP phosphatase activity were found in dividing cells (peaks at circadian times [CT] 00 and 12). The peak observed at CT 00-03 disappeared when the cells had ceased dividing, a result that suggests that it might be regulated by the cell division cycle. NAD kinase activity displayed unimodal circadian rhythms (peak at CT 12) in dividing cells, which persisted with the same phase after the culture entered the stationary phase of growth. Results are discussed with reference to a model (K. Goto, D. L. Laval-Martin, and L. N. Edmunds, Jr., 1985, Science 228, 1284-1288) in which we have proposed that the Ca2(+)-transport system, Ca2+, calmodulin, NAD kinase, and NADP phosphatase could represent clock "gears" that might constitute a self-sustained circadian oscillating loop.

Animals↗
PubMed1975

NAD- and NADP-dependent 7alpha-hydroxysteroid dehydrogenases from bacteroides fragilis.

Twenty strains of Bacteroides fragilis were screened for hydroxysteroid oxidoreductase activity in cell-free preparations. Eighteen strains were shown to contain NAD-dependent 7alpha-hydroxysteroid dehydrogenase. Sixteen of the strains containing the NAD-dependent enzyme also contained NADP-depedent 7alpha-hydroxysteroid dehydrogenase, but invariably in lesser amounts. A strain particulary rich in both 7alpha-hydroxysteroid dehydrogenase activities was selected for further study. Measurement of activity as a function of pH revealed a fairly sharp optimal activity range of 9.5--10.0 for the NAD-dependent enzyme and a broad flat optimal range of 7.0--9.0 for the NADP-dependent enzyme. Michaelis constants for trihydroxy-bile acids for the NAD-dependent enzyme were in the range of 0.32--0.34 mM, whereas dihydroxy-bile acids gave a Km of 0.1 mM. Thin-layer chromatography studies on the oxidation product of 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid) by the dehydrogenase revealed a band corresponding to that of synthetic 3alpha-hydroxy, 7-keto-5beta-cholanoic acid. Similarly the oxidation product of chenodeoxycholic acid by both 7alpha-hydroxysteroid dehydrogenase and commercially available 3alpha-hy-droxysteroid dehydrogenase revealed a band corresponding to that of synthetic 3,7-diketo-5beta-cholanoic acid. Neither of these two oxidation products could be distinguished from those by the Escherichia coli dehydrogenase oxidation previously reported. Disc-gel electrophoresis of a cell-free lyophilized preparation indicated one active band for NAD-dependent activity of mobility similar to that for the NADP-dependent E. coli enzyme. The NADP-dependent dehydrogenase was unstable and rapidly lost activity after polyacylamide disc-gel electrophoresis, ultracentrifugation, freezing on refrigeration at 4 degrees C. No 3 alpha- or 12alpha-oriented oxidoreductase activity was demonstrated in any of the strains examined.

Bacteroides↗