Reduction Of Dihydroxyacetone Phosphate Research Articles

GlcA-mediated glycerol-3-phosphate synthesis contributes to the oxidation resistance of Aspergillus fumigatus via decreasing the cellular ROS

Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.

The Organization of Active Site Side Chains of Glycerol-3-phosphate Dehydrogenase Promotes Efficient Enzyme Catalysis and Rescue of Variant Enzymes.

A comparison of the values of kcat/Km for reduction of dihydroxyacetone phosphate (DHAP) by NADH catalyzed by wild type and K120A/R269A variant glycerol-3-phosphate dehydrogenase from human liver (hlGPDH) shows that the transition state for enzyme-catalyzed hydride transfer is stabilized by 12.0 kcal/mol by interactions with the cationic K120 and R269 side chains. The transition state for the K120A/R269A variant-catalyzed reduction of DHAP is stabilized by 1.0 and 3.8 kcal/mol for reactions in the presence of 1.0 M EtNH3+ and guanidinium cation (Gua+), respectively, and by 7.5 kcal/mol for reactions in the presence of a mixture of each cation at 1.0 M, so that the transition state stabilization by the ternary E·EtNH3+·Gua+ complex is 2.8 kcal/mol greater than the sum of stabilization by the respective binary complexes. This shows that there is cooperativity between the paired activators in transition state stabilization. The effective molarities (EMs) of ∼50 M determined for the K120A and R269A side chains are ≪106 M, the EM for entropically controlled reactions. The unusually efficient rescue of the activity of hlGPDH-catalyzed reactions by the HPi/Gua+ pair and by the Gua+/EtNH3+ activator pair is due to stabilizing interactions between the protein and the activator pieces that organize the K120 and R269 side chains at the active site. This “preorganization” of side chains promotes effective catalysis by hlGPDH and many other enzymes. The role of the highly conserved network of side chains, which include Q295, R269, N270, N205, T264, K204, D260, and K120, in catalysis is discussed.

Human Glycerol 3-Phosphate Dehydrogenase: X-ray Crystal Structures That Guide the Interpretation of Mutagenesis Studies.

Human liver glycerol 3-phosphate dehydrogenase ( hlGPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol 3-phosphate, using the binding energy associated with the nonreacting phosphodianion of the substrate to properly orient the enzyme-substrate complex within the active site. Herein, we report the crystal structures for unliganded, binary E·NAD, and ternary E·NAD·DHAP complexes of wild type hlGPDH, illustrating a new position of DHAP, and probe the kinetics of multiple mutant enzymes with natural and truncated substrates. Mutation of Lys120, which is positioned to donate a proton to the carbonyl of DHAP, results in similar increases in the activation barrier to hlGPDH-catlyzed reduction of DHAP and to phosphite dianion-activated reduction of glycolaldehyde, illustrating that these transition states show similar interactions with the cationic K120 side chain. The K120A mutation results in a 5.3 kcal/mol transition state destabilization, and 3.0 kcal/mol of the lost transition state stabilization is rescued by 1.0 M ethylammonium cation. The 6.5 kcal/mol increase in the activation barrier observed for the D260G mutant hlGPDH-catalyzed reaction represents a 3.5 kcal/mol weakening of transition state stabilization by the K120A side chain and a 3.0 kcal/mol weakening of the interactions with other residues. The interactions, at the enzyme active site, between the K120 side chain and the Q295 and R269 side chains were likewise examined by double-mutant analyses. These results provide strong evidence that the enzyme rate acceleration is due mainly or exclusively to transition state stabilization by electrostatic interactions with polar amino acid side chains.

A cytosolic NAD+-dependent GPDH from maize (ZmGPDH1) is involved in conferring salt and osmotic stress tolerance

BackgroundPlant glycerol-3-phosphate dehydrogenase (GPDH) catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to produce glycerol-3-phosphate (G-3-P), and plays a key role in glycerolipid metabolism as well as stress responses.ResultsIn this study, we report the cloning, enzymatic and physiological characterization of a cytosolic NAD+-dependent GPDH from maize. The prokaryotic expression of ZmGPDH1 in E.coli showed that the enzyme encoded by ZmGPDH1 was capable of catalyzing the reduction of DHAP in the presence of NADH. The functional complementation analysis revealed that ZmGPDH1 was able to restore the production of glycerol-3-phosphate and glycerol in AtGPDHc-deficient mutants. Furthermore, overexpression of ZmGPDH1 remarkably enhanced the tolerance of Arabidopsis to salinity/osmotic stress by enhancing the glycerol production, the antioxidant enzymes activities (SOD, CAT, APX) and by maintaining the cellular redox homeostasis (NADH/NAD+, ASA/DHA, GSH/GSSG). ZmGPDH1 OE Arabidopsis plants also exhibited reduced leaf water loss and stomatal aperture under salt and osmotic stresses. Quantitative real-time RT-PCR analyses revealed that overexpression of ZmGPDH1 promoted the transcripts accumulation of genes involved in cellular redox homeostasis and ROS-scavenging system.ConclusionsTogether, these data suggested that ZmGPDH1 is involved in conferring salinity and osmotic tolerance in Arabidopsis through modulation of glycerol synthesis, stomatal closure, cellular redox and ROS homeostasis.

A multidomain enzyme, with glycerol-3-phosphate dehydrogenase and phosphatase activities, is involved in a chloroplastic pathway for glycerol synthesis in Chlamydomonas reinhardtii.

Understanding the unique features of algal metabolism may be necessary to realize the full potential of algae as feedstock for the production of biofuels and biomaterials. Under nitrogen deprivation, the green alga C.reinhardtii showed substantial triacylglycerol (TAG) accumulation and up-regulation of a gene, GPD2, encoding a multidomain enzyme with a putative phosphoserine phosphatase (PSP) motif fused to glycerol-3-phosphate dehydrogenase (GPD) domains. Canonical GPD enzymes catalyze the synthesis of glycerol-3-phosphate (G3P) by reduction of dihydroxyacetone phosphate (DHAP). G3P forms the backbone of TAGs and membrane glycerolipids and it can be dephosphorylated to yield glycerol, an osmotic stabilizer and compatible solute under hypertonic stress. Recombinant Chlamydomonas GPD2 showed both reductase and phosphatase activities invitro and it can work as a bifunctional enzyme capable of synthesizing glycerol directly from DHAP. In addition, GPD2 and a gene encoding glycerol kinase were up-regulated in Chlamydomonas cells exposed to high salinity. RNA-mediated silencing of GPD2 revealed that the multidomain enzyme was required for TAG accumulation under nitrogen deprivation and for glycerol synthesis under high salinity. Moreover, a GPD2-mCherry fusion protein was found to localize to the chloroplast, supporting the existence of a GPD2-dependent plastid pathway for the rapid synthesis of glycerol in response to hyperosmotic stress. We hypothesize that the reductase and phosphatase activities of PSP-GPD multidomain enzymes may be modulated by post-translational modifications/mechanisms, allowing them to synthesize primarily G3P or glycerol depending on environmental conditions and/or metabolic demands in algal species of the core Chlorophytes.

Enzyme Architecture: Self-Assembly of Enzyme and Substrate Pieces of Glycerol-3-Phosphate Dehydrogenase into a Robust Catalyst of Hydride Transfer.

The stabilization of the transition state for hlGPDH-catalyzed reduction of DHAP due to the action of the phosphodianion of DHAP and the cationic side chain of R269 is between 12.4 and 17 kcal/mol. The R269A mutation of glycerol-3-phosphate dehydrogenase (hlGPDH) results in a 9.1 kcal/mol destabilization of the transition state for enzyme-catalyzed reduction of dihydroxyacetone phosphate (DHAP) by NADH, and there is a 6.7 kcal/mol stabilization of this transition state by 1.0 M guanidine cation (Gua+) [J. Am. Chem. Soc.2015, 137, 5312–5315]. The R269A mutant shows no detectable activity toward reduction of glycolaldehyde (GA), or activation of this reaction by 30 mM HPO32–. We report the unprecedented self-assembly of R269A hlGPDH, dianions (X2– = FPO32–, HPO32–, or SO42–), Gua+ and GA into a functioning catalyst of the reduction of GA, and fourth-order reaction rate constants kcat/KGAKXKGua. The linear logarithmic correlation (slope = 1.0) between values of kcat/KGAKX for dianion activation of wildtype hlGPDH-catalyzed reduction of GA and kcat/KGAKXKGua shows that the electrostatic interaction between exogenous dianions and the side chain of R269 is not significantly perturbed by cutting hlGPDH into R269A and Gua+ pieces. The advantage for connection of hlGPDH (R269A mutant + Gua+) and substrate pieces (GA + HPi) pieces, (ΔGS‡)HPi+E+Gua = 5.6 kcal/mol, is nearly equal to the sum of the advantage to connection of the substrate pieces, (ΔGS‡)GA+HPi = 3.3 kcal/mol, for wildtype hlGPDH-catalyzed reaction of GA + HPi, and for connection of the enzyme pieces, (ΔGS‡)E+Gua = 2.4 kcal/mol, for Gua+ activation of the R269A hlGPDH-catalyzed reaction of DHAP.

The Complex Role of Asn270 in Dianion Activation of Glycerol 3‐Phosphate Dehydrogenase‐Catalyzed Hydride Transfer

We are working to understand the mechanism for the strong phosphite dianion (HPi) activation of proton transfer reactions catalyzed by triosephosphate isomerase (TIM), decarboxylation reactions catalyzed by orotidine monophosphate decarboxylase (OMPDC), and hydride transfer reactions catalyzed by glycerol 3‐phosphate dehydrogenase (GPDH). The phosphodianion of substrate dihydroxyacetone phosphate (DHAP) for GPDH provides 11 kcal/mol stabilization of the transition state for hydride transfer. Eight kcal/mol of this stabilization is observed as HPi activation of GPDH for catalysis of the reduction of glycolaldehyde (GA). The X‐ray crystal structure of the nonproductive complex between GPDH, DHAP and NAD+ shows interactions of Arg269 and Asn270 with the substrate phosphodianion. The R269A mutation of GPDH results in a large 9.1 kcal/mol destabilization of the transition state for enzyme‐catalyzed reduction of DHAP from elimination of the enzyme‐substrate ion‐pair; and 6.7 kcal/mol of this stabilization is recovered when the reaction is carried out in the presence of 1.0 M guanidinium cation.1 Similar effects were reported for K12G and R235A mutations of TIM2 and OMPDC,3 respectively, consistent with a similar architecture of the dianion activation sites for these enzymes. However, the following effects of N270A and R269A/N270A mutations require an unexpected greater mechanistic complexity for dianion activation of GPDH, compared with OMPDC and TIM. 1.) The 5.6 kcal/mol effect from N270A mutation on transition state stability is more than twice as large as that predicted for the deleted phosphodianion‐amide interaction. 2.) The effects of consecutive R269A and N270A mutations depend strongly on the order of these mutations. 3.) The N270A mutation results in a surprising 40‐fold increase in the second‐order rate constant for GPDH‐catalyzed reduction of GA, and causes HPi to change from a strong activator to a weak inhibitor of this GPDH‐catalyzed reaction. These large and complex changes in the activation barriers for hydride transfer show that the amide side chain of Asn270 functions as a “linchpin” in maintaining the structural integrity of the dianion activation site for GPDH.

Abstract 18586: GPD1-L Inhibits the Cardiac Sodium Channel Nav1.5 by decreasing SIRTUIN1 Activity

GPD1-L is a cytosolic protein that catalyzes the reduction of dihydroxyacetone phosphate + NADH into glycerol 3-phosphate + NAD+. Mutations in GPD1-L have been linked to Brugada syndrome and sudden infant death syndrome. GPD1-L regulates the cardiac Na+ channel Nav1.5 but the mechanism is not fully understood. We have recently shown that SIRTUIN1 (SIRT1), a NAD+ dependent deacetylase, increases Na+ current (INa) by deacetylating Nav1.5 and increasing cell surface expression. We therefore tested the hypothesis that GPD1-L mutations alter Nav1.5 activity and INa through a SIRT1-dependent mechanism, and that GPD1L’s catalytic function is an essential component of this regulation. Whole cell patch clamp studies in HEK cells demonstrated that a decrease of GPD1-L expression by siRNA knockdown or catalytic function by mutagenesis increases INa, while overexpression of the A280V Brugada syndrome GPD1-L mutation decreased INa. Moreover, catalytic deficient GPD1-L increased and A280V GPD1-L decreased cell surface expression of Nav1.5. Co-immunoprecipitation studies showed that GPD1-L binds to SIRT1 (Figure A). In addition, catalytically inactive GPD1-L increased and A280V GPD1-L decreased SIRT1deacetylase activity (Figure B). In summary, our evidence supports the hypothesis that GPD1-L functions as a negative regulator of the cardiac Na channel Nav1.5 by interacting with SIRT1 and diminishing its activity; the A280V GPD1-L mutation enhances that inhibition, leading to Brugada syndrome.

Glycerol-3-phosphate and systemic immunity

Glycerol-3-phosphate (G3P), a conserved three-carbon sugar, is an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis. G3P can be derived via the glycerol kinase-mediated phosphorylation of glycerol or G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate. Previously, we showed G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Inoculation of Arabidopsis with C. higginsianum correlated with an increase in G3P levels and a concomitant decrease in glycerol levels in the host. Plants impaired in GLY1 encoded G3Pdh accumulated reduced levels of G3P after pathogen inoculation and showed enhanced susceptibility to C. higginsianum. Recently, we showed that G3P is also a potent inducer of systemic acquired resistance (SAR) in plants. SAR is initiated after a localized infection and confers whole-plant immunity to secondary infections. SAR involves generation of a signal at the site of primary infection, which travels throughout the plants and alerts the un-infected distal portions of the plant against secondary infections. Plants unable to synthesize G3P are defective in SAR and exogenous G3P complements this defect. Exogenous G3P also induces SAR in the absence of a primary pathogen. Radioactive tracer experiments show that a G3P derivative is translocated to distal tissues and this requires the lipid transfer protein, DIR1. Conversely, G3P is required for the translocation of DIR1 to distal tissues. Together, these observations suggest that the cooperative interaction of DIR1 and G3P mediates the induction of SAR in plants.

Green Tea Catechins: Inhibitors of Glycerol-3-Phosphate Dehydrogenase

Green tea catechins, especially (-)-epigallocatechin-3-gallate (EGCG), are known to regulate obesity and fat accumulation. We performed a kinetic analysis in a cell-free system to determine the mode of inhibition of glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) by EGCG. GPDH catalyzes the beta-nicotinamide adenine dinucleotide (NADH)-dependent reduction of dihydroxyacetone phosphate (DHAP) to yield glycerol-3-phosphate, which serves as one of the major precursors of triacylglycerols. We found that EGCG dose-dependently inhibited GPDH activity at a concentration of approximately 20 muM for 50 % inhibition. The IC (50) values of other green tea catechins, such as (-)-epicatechin, (-)-epicatechin-3-gallate, and (-)-epigallocatechin, were all above 100 microM. This suggests a catechin type-dependent effect. Based on double-reciprocal plots of the kinetic data, EGCG was a noncompetitive inhibitor of the GPDH substrates, NADH and DHAP, with respective inhibition constants (Ki) of 18 and 31 microM. Results of this study possibly support previous studies that EGCG mediates fat content.

Cardiac Metabolic State and Brugada Syndrome

See related article, pages 737–745 Brugada syndrome (BrS) is an inherited cardiac disorder with variable ECG features characteristic of right bundle branch bloc (RBBB) and ST segment elevation in the right precordial leads (V1-V3) at rest, which was first described by the Brugada brothers as a clinical entity with a genetic background.1 In Southeast Asia, BrS causes a high risk of sudden unexplained nocturnal death syndrome (SUNDS) in young adults.2,3 In genetically determined forms of BrS, a number of mutations in the SCN5A gene located on chromosome 3p21-23 have been implicated in BrS and account for 20% to 30% of BrS in genotyped families.4 More recently, mutations in other genes such as those coding for glycerol-3-phosphate dehydrogenase 1-like (GPD1-L),5 the α-subunit of the Cav1.2 L-type calcium channel ( CACNA1C ), the β2 calcium channel regulatory subunit ( CACNB2 ),6,7 and the hERG potassium channel ( KCNH2 )8 have been shown to be associated with BrS clinical phenotypes. Mutations in the SCN5A gene are associated with loss-of-function caused by trafficking defects, truncated sodium channel proteins, or changes in the biophysical properties of sodium channels. Loss-of-function mutations of the Cav1.2 L-type calcium channel and its regulatory subunit, which are encoded by CACNA1C and CACNB2 , respectively (A39V- and G490R/ CACNA1C and S481L/ CACNB2 ), have been reported to be associated with a BrS phenotype combined with shorter QT intervals. Two gain-of-function mutations in the KCNH2 (hERG) of the cardiac potassium channel (G873S and N985S) have been reported in patients with BrS syndrome. Glycerol-3-phosphate dehydrogenase (GPD) catalyzes the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate. GPD uses nicotinamide adenine dinucleotide (NADH) as a cofactor for this …

The common metabolite glycerol-3-phosphate is a novel regulator of plant defense signaling

Conversion of glycerol to glycerol-3-phosphate (G3P) is one of the highly conserved steps of glycerol metabolism in evolutionary diverse organisms. In plants, G3P is produced either via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or via G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP). We have recently shown that G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Since a mutation in the GLY1-encoded G3Pdh conferred more susceptibility compared to a mutation in the GLI1-encoded GK, we proposed that GLY1 is the major contributor of the total G3P pool that participates in defense against C. higginsianum.

Glyceroneogenesis Is the Dominant Pathway for Triglyceride Glycerol Synthesis in Vivo in the Rat

Triglyceride synthesis in mammalian tissues requires glycerol 3-phosphate as the source of triglyceride glycerol. In this study the relative contribution of glyceroneogenesis and glycolysis to triglyceride glycerol synthesis was quantified in vivo in adipose tissue, skeletal muscle, and liver of the rat in response to a chow diet (controls), 48-h fast, and lipogenic (high sucrose) diet. The rate of glyceroneogenesis was quantified using the tritium ([(3)H(2)]O) labeling of body water, and the contribution of glucose, via glycolysis, was determined using a [U-(14)C]glucose tracer. In epididymal and mesenteric adipose tissue of control rats, glyceroneogenesis accounted for approximately 90% of triglyceride glycerol synthesis. Fasting for 48 h did not alter glyceroneogenesis in adipose tissue, whereas the contribution of glucose was negligible. In response to sucrose feeding, the synthesis of triglyceride glycerol via both glyceroneogenesis and glycolysis nearly doubled (versus controls); however, glyceroneogenesis remained quantitatively higher as compared with the contribution of glucose. Enhancement of triglyceride-fatty acid cycling by epinephrine infusion resulted in a higher rate of glyceroneogenesis in adipose tissue, as compared with controls, whereas the contribution of glucose via glycolysis was not measurable. Glyceroneogenesis provided the majority of triglyceride glycerol in the gastrocnemius and soleus. In the liver the fractional contribution of glyceroneogenesis remained constant (approximately 60%) under all conditions and was higher than that of glucose. Thus, glyceroneogenesis, in contrast to glucose, via glycolysis, is quantitatively the predominant source of triglyceride glycerol in adipose tissue, skeletal muscle, and liver of the rat during fasting and high sucrose feeding.

A Substrate in Pieces: Allosteric Activation of Glycerol 3-Phosphate Dehydrogenase (NAD+) by Phosphite Dianion

The ratio of the second-order rate constants for reduction of dihydroxyacetone phosphate (DHAP) and of the neutral truncated substrate glycolaldehyde (GLY) by glycerol 3-phosphate dehydrogenase (NAD (+), GPDH) saturated with NADH is (1.0 x 10 (6) M (-1) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 1.1 x 10 (8), which was used to calculate an intrinsic phosphate binding energy of at least 11.0 kcal/mol. Phosphite dianion binds very weakly to GPDH ( K d > 0.1 M), but the bound dianion strongly activates GLY toward enzyme-catalyzed reduction by NADH. Thus, the large intrinsic phosphite binding energy is expressed only at the transition state for the GPDH-catalyzed reaction. The ratio of rate constants for the phosphite-activated and the unactivated GPDH-catalyzed reduction of GLY by NADH is (4300 M (-2) s (-1))/(8.7 x 10 (-3) M (-1) s (-1)) = 5 x 10 (5) M (-1), which was used to calculate an intrinsic phosphite binding energy of -7.7 kcal/mol for the association of phosphite dianion with the transition state complex for the GPDH-catalyzed reduction of GLY. Phosphite dianion has now been shown to activate bound substrates for enzyme-catalyzed proton transfer, decarboxylation, hydride transfer, and phosphoryl transfer reactions. Structural data provide strong evidence that enzymic activation by the binding of phosphite dianion occurs at a modular active site featuring (1) a binding pocket complementary to the reactive substrate fragment which contains all the active site residues needed to catalyze the reaction of the substrate piece or of the whole substrate and (2) a phosphate/phosphite dianion binding pocket that is completed by the movement of flexible protein loop(s) to surround the nonreacting oxydianion. We propose that loop motion and associated protein conformational changes that accompany the binding of phosphite dianion and/or phosphodianion substrates lead to encapsulation of the substrate and/or its pieces in the protein interior, and to placement of the active site residues in positions where they provide optimal stabilization of the transition state for the catalyzed reaction.

Isolation and properties of cytoplasmic alpha-glycerol 3-phosphate dehydrogenase from the pectoral muscle of the fruit bat, Eidolon helvum.

Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.

Glycerol-3-phosphate acquisition in spirochetes: distribution and biological activity of glycerophosphodiester phosphodiesterase (GlpQ) among Borrelia species.

Relapsing-fever spirochetes achieve high cell densities (>10(8)/ml) in their host's blood, while Lyme disease spirochetes do not (<10(5)/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood. Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B. hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids. Sequence analysis of B. hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B. burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.

Kinetic study of sn-glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1.

A gene having high sequence homology (45-49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94-96 degrees C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H-dependent dihydroxyacetone phosphate reduction and NAD(+)-dependent glycerol-1-phosphate (Gro1P) oxidation. NADP(+)-dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)(+) acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi-bi mechanism.

Purification and characterization of cytosolic glycerol-3-phosphate dehydrogenase from skeletal muscle of jerboa (Jaculus orientalis).

Cytosolic glycerol-3-phosphate dehydrogenase was purified from jerboa (Jaculus orientalis) skeletal muscle and its physical and kinetic properties investigated. The purification method consisted of a multi-step procedure and this procedure is presented. The specific activity of the purified enzyme is 53.6 U/mg of protein, representing a 77-fold increase in specific activity. The apparent Michaelis constant (Km) for dihydroxyacetone is 137.39 (+/- 25.56) microM whereas the Km for glycerol-3-phosphate is 468.66 (+/- 27.59) microM. The kinetic mechanism of purified enzyme is 'ordered Bi-Bi' and this result is confirmed by the product inhibition pattern. Under the conditions of assay, the pH optimum occurs at pH 7.7 for the reduction of dihydroxyacetone phosphate and at pH 9.0 for glycerol-3-phosphate oxidation. In the direction of dihydroxyacetone phosphate, the optimal temperature is 35 degrees C. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 33,000 (+/- 1000), whereas non-denaturing polyacrylamide gel yields a molecular weight of 72,000 (+/- 2000), suggesting that the enzyme may exist as a dimer. A polyclonal antiserum raised against the purified enzyme was used to localize the enzyme in different jerboa tissues by Western blot method. The purified enzyme is sensitive to N-ethylmaleimide, and incubation of the enzyme with 20 mM N-ethylmaleimide resulted in a complete loss of catalytic activity. The purified enzyme is inhibited by several metal ions including Zn2+ and by 2,4-dichlorophenoxyacetic acid.

Molecular and biochemical characterizations of a plastidic glycerol-3-phosphate dehydrogenase from Arabidopsis

Glycerol-3-phosphate dehydrogenase (NAD +-G-3-P oxidoreductase, EC 1.1.1.8) (GPDH) catalyses the reduction of dihydroxyacetone phosphate (DHAP) to form glycerol-3-phosphate (G-3-P), which is essential for phospholipid synthesis through both the prokaryotic and the eukaryotic glycerolipid pathway. A cDNA ( AtGPDHp) encoding for the plastidic GPDH from Arabidopsis thaliana was cloned. The predicted AtGPDHp has in its amino-terminal region a chloroplast targeting signal peptide-like sequence. Transgenic tobacco plants with a chimeric green fluorescent protein (GFP) construct showed that the N-terminal peptide of AtGPDHp targeted GFP into chloroplasts. The functional identity of AtGPDHp was experimentally confirmed by expression of AtGPDHp in an Escherichia coli ( gpsA) mutant auxotrophic of G-3-P. The K m value and V max of a purified recombinant AtGPDHp for DHAP were 12.8 μM and 3.2 μkat·min –1·mg –1 protein, respectively. Northern blot experiments indicated that AtGPDHp gene is expressed in all tissues examined, but its transcript is particularly abundant in developing siliques and flowers. This study represents the first molecular cloning and expression analysis of a plastidic GPDH in plants.

Site-Specific Hydrogen Isotope Fractionation in the Biosynthesis of Glycerol

The nuclear magnetic resonance study of site-specific natural isotope fractionation (SNIF-NMR) produced in the glycolytic conversion of glucose into ethanol and glycerol provides isotopic transfer coefficients, aij, which relate sites in the products to sites j in the reactants. The isotopic connection between the carbon-bound hydrogens of glycerol and those of glucose and water in fermentation reactions carried out with Saccharomyces cerevisiae has been investigated. The aij coefficients provide mechanistic information on the genealogy of the glycerol hydrogens, on the relative rates of triose phosphate isomerization and reduction of dihydroxyacetone phosphate into glycerol 3-phosphate, on the stereospecificity of the reduction, on the percentages of intra- and intermolecular hydrogen transfer occurring in the course of the reaction, and on the order of magnitude of the active overall kinetic and thermodynamic isotope effects. Thus a close connection is determined between sites 3 pro-R of glycerol (stereospecific numbering) and H6 pro-R and to a lesser extent H2 of glucose and between site 3 pro-S of glycerol and H6 pro-S and H1 of glucose. Moreover site 2 of glycerol, which exhibits a strong correlation with water is also partly connected with glucose. Possible changes in the values of the isotopic transfer coefficients, as a function of the composition of the medium or of the environmental conditions, enable mechanistic perturbations such as variations in the percentage of intermolecular transfers to be detected. Analyzed in terms of stereospecificity of the reduction step the isotopic results provide a direct chemical shift assignment of the enantiomeric pairs (1S, 3R) and (1R, 3S) of glycerol triacetate. The influence of added bisulfite, which strongly increases the yield in glycerol is estimated. The isotopic characterization of the bioconversion producing both ethanol and glycerol has been extended to the determination of the carbon isotopic parameters by isotope ratio mass spectrometry. Although it usually occurs as a by-product of the fermentation, glycerol can be considered as a useful complementary probe for characterizing the glycolytic pathway and for inferring various properties of the carbohydrate precursors.