Stereoselectivity of fructose-1,6-bisphosphate aldolase in Thermus caldophilus

It was recently established that fructose-1,6-bisphosphate (FBP) aldolase (FBA) and tagatose-1,6-bisphosphate (TBP) aldolase (TBA), two class II aldolases, are highly specific for the diastereoselective synthesis of FBP and TBP from glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), respectively. In this paper, we report on a FBA from the thermophile Thermus caldophilus GK24 (Tca) that produces both FBP and TBP from C(3) substrates. Moreover, the FBP:TBP ratio could be adjusted by manipulating the concentrations of G3P and DHAP. This is the first native FBA known to show dual diastereoselectivity among the FBAs and TBAs characterized thus far. To explain the behavior of this enzyme, the X-ray crystal structure of the Tca FBA in complex with DHAP was determined at 2.2A resolution. It appears that as a result of alteration of five G3P binding residues, the substrate binding cavity of Tca FBA has a greater volume than those in the Escherichia coli FBA-phosphoglycolohydroxamate (PGH) and TBA-PGH complexes. We suggest that this steric difference underlies the difference in the diastereoselectivities of these class II aldolases.     

One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis

Rv0363c (fba), encoding Class II fructose-bisphosphate aldolase (FBA), is one of the potential drug targets identified in our laboratory based on minimal gene set concept. The wild-type enzyme overproduction in E. coli had been reported. However, the purification procedure was relatively tedious and the yield was low. In this study, five histidine codons were introduced into the 3′ end of the amplified fba fragments. The expressed C-terminal histidine-tagged Class II FBA was produced in E. coli BL21 (DE3) and easily purified using immobilized metal affinity chromatography. The purified his-tagged FBA was characterized. Its biochemical properties were compared to the non-his-tagged enzyme purified according to the previous report. Both FBAs have similar characteristics such as native/subunit molecular mass, kinetic parameters, and temperature/pH optima and stability. The C-terminal his-tagged FBA can be a surrogate for the native enzyme and used for screening of inhibitors of FBA. This developed expression system will pave the way for high-throughput screening and crystallization studies. Moreover, in this study, a colorimetric FBA assay has been simplified to facilitate the mass screening of inhibitor of FBA.     

Inactivation of Fructose-1,6-Bisphosphate Aldolase Prevents Optimal Co-catabolism of Glycolytic and Gluconeogenic Carbon Substrates in Mycobacterium tuberculosis

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb''s ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb''s ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb''s FBA in vivo.     

Molecular modeling,dynamics studies and virtual screening of Fructose 1, 6 biphosphate aldolase-II in community acquired- methicillin resistant Staphylococcus aureus (CA-MRSA)

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged as a nosocomial pathogen to the community which commonly causes skin and soft-tissue infections (SSTIs). This strain (MW2) has now become resistant to the most of the beta-lactam antibiotics; therefore it is the urgent need to identify the novel drug targets. Recently fructose 1,6 biphosphate aldolase-II (FBA) has been identified as potential drug target in CA-MRSA. The FBA catalyses the retro-ketolic cleavage of fructose-1,6-bisphosphate (FBP) to yield dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) in glycolytic pathway. In the present research work the 3D structure of FBA was predicted using the homology modeling method followed by validation. The molecular dynamics simulation (MDS) of the predicted model was carried out using the 2000 ps time scale and 1000000 steps. The MDS results suggest that the modeled structure is stable. The predicted model of FBA was used for virtual screening against the NCI diversity subset-II ligand databases which contain 1364 compounds. Based on the docking energy scores, it was found that top four ligands i.e. ZINC01690699, ZINC13154304, ZINC29590257 and ZINC29590259 were having lower energy scores which reveal higher binding affinity towards the active site of FBA. These ligands might act as potent inhibitors for the FBA so that the menace of antimicrobial resistance in CA-MRSA can be conquered. However, pharmacological studies are required to confirm the inhibitory activity of these ligands against the FBA in CA-MRSA.     

Regulation of beta-D-galactosidase synthesis in Candida pseudotropicalis.

Regulation of lactose (beta-D-galactosidase) synthesis in the lactose-utilizing yeast Candida pseudotropicalis was studied. The enzyme was inducible by lactose and galactose. When grown on these sugars the enzyme level of the yeast was 20 times or higher than when grown on glycerol. The Km and optimal pH were similar for the lactase induced either by lactose or galactose. The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside by the lactase was inhibited by galactose and several analogs and galactosides, but not by glucose. Lactose uptake activity observed in lactose-grown cells was very reduced in cells grown on glucose or galactose. Glucose repressed the induction of lactase, but not the metabolic system for galactose utilization. In continuous culture on lactose medium at dilution rates below 0.2 h-1 the specific lactase activity was higher than in batch cultures and decreased with increases in dilution rate. Lactase was induced by pulses of lactose and galactose in cells growing on glucose, but only at low dilution rates were the steady-state concentration of glucose was very low.     

Characterization of recombinant fructose-1,6-bisphosphate aldolase from <Emphasis Type="Italic">Methylococcus capsulatus</Emphasis> Bath

The gene fba from the thermotolerant obligate methanotroph Methylococcus capsulatus Bath was cloned and expressed in Escherichia coli BL21(DE3). The fructose-1,6-bisphosphate aldolase (FBA) carrying six His on the C-end was purified by affinity metal chelating chromatography. The Mc. capsulatus FBA is a hexameric enzyme (240 kDa) that is activated by Co²⁺ and inhibited by EDTA. The enzyme displays low K _m to fructose-1,6-bisphosphate (FBP) and higher K _m to the substrates of aldol condensation, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. The FBA also catalyzes sedoheptulose-1,7-bisphosphate cleavage. The presence of Co²⁺ in the reaction mixture changes the kinetics of FBP hydrolysis and is accompanied by inhibition of the reaction by 2 mM FBP. Phylogenetically, the Mc. capsulatus enzyme belongs to the type B of class II FBAs showing high identity of translated amino acid sequence with FBAs from autotrophic bacteria. The role of the FBA in metabolism of Mc. capsulatus Bath, which realizes simultaneously three C₁ assimilating pathways (the ribulose monophosphate, the ribulose bisphosphate, and the serine cycles), is discussed.     

Construction of a constitutively expressed homo-fermentative pathway in Lactobacillus brevis

Lactobacillus brevis is a promising lactic acid producing strain that simultaneously utilizes glucose and xylose from lignocellulosic hydrolysate without carbon catabolic repression and inhibition. The production of by-products acetic acid and ethanol has been the major drawback of this strain. Two genes, pfkA (fructose-6-phosphate kinase [PFK]) and fbaA (fructose-1,6-biphosphate aldolase [FBA]), that encode the key enzymes of the EMP/glycolytic pathway from Lactobacillus rhamnosus, were fused to the downstream of the strong promoter P32 and expressed in L. brevis s3f4 as a strategy to minimize the formation of by-products. By expressing the two enzymes, a homo-fermentative pathway for lactic acid production was constructed. The lactic acid yields achieved from glucose in the transformants were 1.12 and 1.16 mol/mol, which is higher than that of the native strain (0.74 mol/mol). However, the lactic acid yield from xylose in the transformants stayed the same as that of the native strain. Enzyme assay indicated that the activity of the foreign protein FBA in the transformants was much higher than that of the native strains, but was ten times lower than that in L. rhamnosus. This result was consistent with the metabolic flux analysis, which indicated that the conversion efficiency of the expressed PFK and FBA was somewhat low. Less than 20 % of the carbons accumulated in the form of fructose-6-phosphate were converted into glyceraldehyde-3-phosphate (GAP) by the expressed PFK and FBA. Metabolic flux analysis also indicated that the enzyme phosphoketolase (XPK) played an important role in splitting the carbon flow from the pentose phosphate pathway to the phosphoketolase pathway. This study suggested that the lactic acid yield of L. brevis could be improved by constructing a homo-fermentative pathway.     

One-step purification and characterization of a fully active histidine-tagged Class II fructose-1,6-bisphosphate aldolase from Mycobacterium tuberculosis

Rv0363c (fba), encoding Class II fructose-bisphosphate aldolase (FBA), is one of the potential drug targets identified in our laboratory based on minimal gene set concept. The wild-type enzyme overproduction in E. coli had been reported. However, the purification procedure was relatively tedious and the yield was low. In this study, five histidine codons were introduced into the 3′ end of the amplified fba fragments. The expressed C-terminal histidine-tagged Class II FBA was produced in E. coli BL21 (DE3) and easily purified using immobilized metal affinity chromatography. The purified his-tagged FBA was characterized. Its biochemical properties were compared to the non-his-tagged enzyme purified according to the previous report. Both FBAs have similar characteristics such as native/subunit molecular mass, kinetic parameters, and temperature/pH optima and stability. The C-terminal his-tagged FBA can be a surrogate for the native enzyme and used for screening of inhibitors of FBA. This developed expression system will pave the way for high-throughput screening and crystallization studies. Moreover, in this study, a colorimetric FBA assay has been simplified to facilitate the mass screening of inhibitor of FBA.     

Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae.

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.     

The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

Studies of metabolism of round spermatids: glucose as unfavorable substrate

The exposure of spermatids to glucose in the absence of pyruvate and lactate resulted in an extremely low energy charge. The adenosine 5'-triphosphate (ATP) level rapidly declined and the fructose 1,6-bisphosphate (FBP) and triose levels increased. These changes were prevented by the addition of pyruvate or lactate. The levels of ATP and FBP were inversely correlated. In cells exposed to glucose, FBP did not flow appreciably through the step of glyceraldehyde 3-phosphate dehydrogenase (GA3PDH). The lactate level did not change. However, when pyruvate or lactate was administered to cells exposed to glucose, the FBP level declined rapidly. This drop was accompanied by a commensurate increase in lactate. In these cells, pyruvate transport was suppressed, and the pyruvate taken up by these cells was mostly oxidized in the tricarboxylic acid (TCA) cycle without its being reduced to lactate. In this case, the ATP level increased, but to a level still lower than existed before exposure to glucose. Furthermore, when kinetic studies on the activity of 6-phosphofructokinase (PFK) were carried out, PFK appeared to be fully activated at intracellular levels of fructose 6-phosphate, ATP and adenosine 5'-monophosphate (AMP). These results indicate that the rate of glucose metabolism in glycolysis depends heavily on the energy charge. In cells exposed to glucose, the sugar does not flow appreciably through the glycolytic pathway due to inhibition of GA3PDH. Moreover, the ATP level cannot be recovered fully from the lowest level by the addition of pyruvate or lactate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Dual Glycolytic Pathways for Fructose Metabolism in Heterofermentative Lactobacillus panis PM1

Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli that use the 6-phosphogluconate/phosphoketolase (6-PG/PK) pathway as their central metabolic pathway and are reportedly unable to grow on fructose as a sole carbon source. We isolated a variant PM1 strain capable of sporadic growth on fructose medium and observed its distinctive characteristics of fructose metabolism. The end product pattern was different from what is expected in typical group III lactobacilli using the 6-PG/PK pathway (i.e., more lactate, less acetate, and no mannitol). In addition, in silico analysis revealed the presence of genes encoding most of critical enzymes in the Embden-Meyerhof (EM) pathway. These observations indicated that fructose was metabolized via two pathways. Fructose metabolism in the PM1 strain was influenced by the activities of two enzymes, triosephosphate isomerase (TPI) and glucose 6-phosphate isomerase (PGI). A lack of TPI resulted in the intracellular accumulation of dihydroxyacetone phosphate (DHAP) in PM1, the toxicity of which caused early growth cessation during fructose fermentation. The activity of PGI was enhanced by the presence of glyceraldehyde 3-phosphate (GAP), which allowed additional fructose to enter into the 6-PG/PK pathway to avoid toxicity by DHAP. Exogenous TPI gene expression shifted fructose metabolism from heterolactic to homolactic fermentation, indicating that TPI enabled the PM1 strain to mainly use the EM pathway for fructose fermentation. These findings clearly demonstrate that the balance in the accumulation of GAP and DHAP determines the fate of fructose metabolism and the activity of TPI plays a critical role during fructose fermentation via the EM pathway in L. panis PM1.     

The expression of β-galactosidase by Escherichia coli during continuous culture

We have used the technique of continuous culture to study the expression of β-galactosidase in Escherichia coli. In these experiments the cultures were grown on carbon-limited media in which half of the available carbon was supplied as glycerol, glucose, or glucose 6-phosphate, and the other half as lactose. Lactose itself provided the sole source of inducer for the lac operon. The steady-state specific activity of the enzyme passed through a maximal value as a function of dilution rate. Moreover, the rate at which activity was maximal (0.40 h^?1) and the observed specific activity of the enzyme at a given growth rate were found to be identical in each of the three media tested. This result was unexpected, since the steady-state specific activity can be shown to be equal to the differential rate of enzyme synthesis, and since it is known that glycerol, glucose, and glucose-6-P-cause different degrees of catabolite repression in batch culture. The differential rate of β-galactosidase synthesis was an apparently linear function of the rate of lactose utilization per milligram protein regardless of the composition of the input medium. That is, it is independent of the rate of metabolism of substrates other than lactose which are concurrently being utilized and the enzyme level appears to be matched to the metabolic requirement for it. If this relationship is taken to indicate the existence of a fundamental control mechanism, it may represent a form of attenuation of the rate of β-galactosidase synthesis which is independent of cyclic AMP levels.     

Gluconeogenic mutations in Pseudomonas aeruginosa: genetic linkage between fructose-bisphosphate aldolase and phosphoglycerate kinase

Mutants of mucoid Pseudomonas aeruginosa defective in fructose-bisphosphate aldolase (FBA), NADP-linked glyceraldehyde-3-phosphate dehydrogenase (GAP) or 3-phosphoglycerate kinase (PGK) were unable to grow on gluconeogenic precursors like glutamate, succinate or lactate. The gap and pgk mutants could grow on glucose, gluconate or glycerol, but fba mutants could not. This suggests that the metabolism of glucose or gluconate does not require either PGK or NADP-linked GAP but does require the operation of the aldolase-catalysed step. For gluconeogenesis, however, all three steps are essential. Recombinant plasmids carrying genes for FBA, PGK, GAP or phospho-2-keto-3-deoxygluconate aldolase (EDA) activities were constructed from a genomic library of mucoid P. aeruginosa selecting for complementation of deficiency mutations. Analysis of their complementation profile indicated that one group of plasmids carried fba and pgk genes, while another group carried eda, 6-phosphogluconate dehydratase (edd) and glucose-6-phosphate dehydrogenase (zwf) genes. The gap gene was not linked to any of these markers. Partial restoration of FBA activity in spontaneous revertants of Fba- mutants was accompanied by a concomitant loss of PGK activity. These experiments indicate a linkage between the fba and pgk genes on the P. aeruginosa chromosome.     

Energy recycling by lactate efflux in growing and nongrowing cells of Streptococcus cremoris.

Streptococcus cremoris was grown in pH-regulated batch and continuous cultures with lactose as the energy source. During growth the magnitude and composition of the electrochemical proton gradient and the lactate concentration gradient were determined. The upper limit of the number of protons translocated with a lactate molecule during lactate excretion (the proton-lactate stoichiometry) was calculated from the magnitudes of the membrane potential, the transmembrane pH difference, and the lactate concentration gradient. In cells growing in continuous culture, a low lactate concentration gradient (an internal lactate concentration of 35 to 45 mM at an external lactate concentration of 25 mM) existed. The cell yield (Ymax lactose) increased with increasing growth pH. In batch culture at pH 6.34, a considerable lactate gradient (more than 60 mV) was present during the early stages of growth. As growth continued, the electrochemical proton gradient did not change significantly (from -100 to -110 mV), but the lactate gradient decreased gradually. The H+-lactate stoichiometry of the excretion process decreased from 1.5 to about 0.9. In nongrowing cells, the magnitude and composition of the electrochemical proton gradient was dependent on the external pH but not on the external lactate concentration (up to 50 mM). The magnitude of the lactate gradient was independent of the external pH but decreased greatly with increasing external lactate concentrations. At very low lactate concentrations, a lactate gradient of 100 mV existed, which decreased to about 40 mV at 50 mM external lactate. As a consequence, the proton-lactate stoichiometry decreased with increasing external concentrations of protons and lactate at pH 7.0 from 1 mM lactate to 1.1 at 50 mM lactate and at pH 5.5 from 1.4 at l mM lactate to 0.7 at 50 mM lactate. The data presented in this paper suggest that a decrease in external pH and an increase in external lactate concentration both result in lower proton-lactate stoichiometry values and therefore in a decrease of the generation of metabolic energy by the end product efflux process.