Enzymatic characterization of 5-methylthioribose 1-phosphate isomerase from Bacillus subtilis

The product of the mtnA gene of Bacillus subtilis catalyzes the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). The catalysis of MtnA is a novel isomerization of an aldose phosphate harboring a phosphate group on the hemiacetal group. This enzyme is distributed widely among bacteria through higher eukaryotes. The isomerase reaction analyzed using the recombinant B. subtilis enzyme showed a Michaelis constant for MTR-1-P of 138 microM, and showed that the maximum velocity of the reaction was 20.4 micromol min(-1) (mg protein)(-1). The optimum reaction temperature and reaction pH were 35 degrees C and 8.1. The activation energy of the reaction was calculated to be 68.7 kJ mol(-1). The enzyme, with a molecular mass of 76 kDa, was composed of two subunits. The equilibrium constant in the reversible isomerase reaction [MTRu-1-P]/[MTR-1-P] was 6. We discuss the possible reaction mechanism.     

RuBisCO-like proteins as the enolase enzyme in the methionine salvage pathway: functional and evolutionary relationships between RuBisCO-like proteins and photosynthetic RuBisCO

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme in the fixation of CO(2) in the Calvin cycle of plants. Many genome projects have revealed that bacteria, including Bacillus subtilis, possess genes for proteins that are similar to the large subunit of RuBisCO. These RuBisCO homologues are called RuBisCO-like proteins (RLPs) because they are not able to catalyse the carboxylase or the oxygenase reactions that are catalysed by photosynthetic RuBisCO. It has been demonstrated that B. subtilis RLP catalyses the 2,3-diketo-5-methylthiopentyl-1-phosphate (DK-MTP-1-P) enolase reaction in the methionine salvage pathway. The structure of DK-MTP-1-P is very similar to that of ribulose-1,5-bisphosphate (RuBP) and the enolase reaction is a part of the reaction catalysed by photosynthetic RuBisCO. In this review, functional and evolutionary relationships between B. subtilis RLP of the methionine salvage pathway, other RLPs, and photosynthetic RuBisCO are discussed. In addition, the fundamental question, 'How has RuBisCO evolved?' is also considered, and evidence is presented that RuBisCOs evolved from RLPs.     

Crystal structure of 5-methylthioribose 1-phosphate isomerase product complex from Bacillus subtilis: implications for catalytic mechanism

The methionine salvage pathway (MSP) plays a crucial role in recycling a sulphahydryl derivative of the nucleoside. Recently, the genes and reactions in MSP from Bacillus subtilis have been identified, where 5-methylthioribose 1-phosphate isomerase (M1Pi) catalyzes a conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P). Herein, we report the crystal structures of B. subtilis M1Pi (Bs-M1Pi) in complex with its product MTRu-1-P, and a sulfate at 2.4 and 2.7 A resolution, respectively. The electron density clearly shows the presence of each compound in the active site. The structural comparison with other homologous proteins explains how the substrate uptake of Bs-M1Pi may be induced by an open/closed transition of the active site. The highly conserved residues at the active site, namely, Cys160 and Asp240 are most likely to be involved in catalysis. The structural analysis sheds light on its catalytic mechanism of M1Pi.     

Kinetic properties of glucose-6-phosphate dehydrogenase from lamb kidney cortex

Glucose-6-phosphate dehydrogenase is the key regulatory enzyme of the pentose phosphate pathway and one of the products of this enzyme; NADPH has a critical role in the defence system against the free radicals. In this study, glucose-6-phosphate dehydrogenase from lamb kidney cortex kinetic properties is examined. The purification procedure is composed of two steps after ultracentrifugation for rapid and easy purification: 2', 5'-ADP Sepharose 4B affinity and DEAE Sepharose Fast Flow anion exchange chromatography. Previously, we used this procedure for the purification of glucose-6-phosphate dehydrogenase from bovine lens. The double reciprocal plots and product inhibition studies showed that the enzyme obeys 'Ordered Bi Bi' mechanism: K(m NADP+)K(m G-6-P) and K(i G-6-P) (dissociation constant of the enzyme--G-6-P complex) were found to be 0.018 +/- 0.002, 0.039 +/- 0.006 and 0.029 +/- 0.005 mM, respectively, by using nonlinear regression analysis. The enzyme was stable at 4 degrees C for a week.     

Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.     

Structure and mechanism of an ADP-glucose phosphorylase from Arabidopsis thaliana

The X-ray crystal structure of the At5g18200.1 protein has been determined to a nominal resolution of 2.30 A. The structure has a histidine triad (HIT)-like fold containing two distinct HIT-like motifs. The sequence of At5g18200.1 indicates a distant family relationship to the Escherichia coli galactose-1-P uridylyltransferase (GalT): the determined structure of the At5g18200.1 protein confirms this relationship. The At5g18200.1 protein does not demonstrate GalT activity but instead catalyzes adenylyl transfer in the reaction of ADP-glucose with various phosphates. The best acceptor among those evaluated is phosphate itself; thus, the At5g18200.1 enzyme appears to be an ADP-glucose phosphorylase. The enzyme catalyzes the exchange of (14)C between ADP-[(14)C]glucose and glucose-1-P in the absence of phosphate. The steady state kinetics of exchange follows the ping-pong bi-bi kinetic mechanism, with a k(cat) of 4.1 s(-)(1) and K(m) values of 1.4 and 83 microM for ADP-[(14)C]glucose and glucose-1-P, respectively, at pH 8.5 and 25 degrees C. The overall reaction of ADP-glucose with phosphate to produce ADP and glucose-1-P follows ping-pong bi-bi steady state kinetics, with a k(cat) of 2.7 s(-)(1) and K(m) values of 6.9 and 90 microM for ADP-glucose and phosphate, respectively, at pH 8.5 and 25 degrees C. The kinetics are consistent with a double-displacement mechanism that involves a covalent adenylyl-enzyme intermediate. The X-ray crystal structure of this intermediate was determined to 1.83 A resolution and shows the AMP group bonded to His(186). The value of K(eq) in the direction of ADP and glucose-1-P formation is 5.0 at pH 7.0 and 25 degrees C in the absence of a divalent metal ion, and it is 40 in the presence of 1 mM MgCl(2).     

Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues

Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-(13)C(2)]-PEP and [3-F]-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and [2,3-(13)C(2)]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E. coli KDO8-P synthase.     

Molecular characterization of a phosphoenolpyruvate carboxylase from a thermophilic cyanobacterium, Synechococcus vulcanus with unusual allosteric properties

A gene for phosphoenolpyruvate carboxylase (PEPC) was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, by screening a genomic DNA library using the coding region of Anacystis nidulans 6301 PEPC as a probe. The S. vulcanus PEPC gene (SvPEPC) had an open reading frame for a polypeptide of 1,011 amino acid residues with a calculated molecular mass of 116.4 kDa. SvPEPC was expressed in E. coli BL21 Codonplus (DE3), using pET32a as a vector. The purified recombinant SvPEPC protein with a tag showed a single band of 120 kDa on SDS-PAGE. The enzyme forms homotetramer as judged by gel filtration. SvPEPC retained full activity even after incubation at 50 degrees C for 60 min or exposure to 0.5 M guanidine-HCl at 30 degrees C for 20 h, being more stable than C4-form PEPC from Zea mays (ZmPEPC(C4)). SvPEPC activity showed a sharp optimum temperature of 42 degrees C at pH 7.5 and an optimum pH of 9.0 at 30 degrees C. The enzyme, unlike most plant PEPCs, was predominantly activated by fructose 1,6-bisphosphate (Fruc-1,6-P(2)), and slightly stimulated by 3-phosphoglycerate (3-PGA), glucose 6-phosphate (Gluc-6-P), glucose 1-phosphate, Glu and Gln. Acetyl-CoA known as a strong activator of most bacterial PEPCs but not of plant PEPCs, showed no effect on the enzyme activity. SvPEPC was more sensitive to the inhibition by Asp at higher pH (9.0) than lower pH (7.0), contrary to Coccochloris peniocystis PEPC and plant PEPCs. I(0.5) for Asp was increased about 2-fold by Gluc-6-P while markedly decreased by Fruc-1,6-P(2), Glu and Gln about 3- to 4-fold. The regulation mechanism of SvPEPC is not readily interpretable by conventional allosteric models.     

Transaldolase of Methanocaldococcus jannaschii

The Methanocaldococcus jannaschii genome contains putative genes for all four nonoxidative pentose phosphate pathway enzymes. Open reading frame (ORF) MJ0960 is a member of the mipB/talC family of 'transaldolase-like' genes, so named because of their similarity to the well-characterized transaldolase B gene family. However, recently, it has been reported that both the mipB and the talC genes from Escherichia coli encode novel enzymes with fructose-6-phosphate aldolase activity, not transaldolase activity (Schürmann and Sprenger 2001). The same study reports that other members of the mipB/talC family appear to encode transaldolases. To confirm the function of MJ0960 and to clarify the presence of a nonoxidative pentose phosphate pathway in M. jannaschii, we have cloned ORF MJ0960 from M. jannaschii genomic DNA and purified the recombinant protein. MJ0960 encodes a transaldolase and displays no fructose-6-phosphate aldolase activity. It etained full activity for 4 h at 80 degrees C, and for 3 weeks at 25 degrees C. Methanocaldococcus jannaschii transaldolase has a maximal velocity (Vmax) of 1.0 +/- 0.2 micromol min(-1) mg(-1) at 25 degrees C, whereas Vmax = 12.0 +/- 0.5 micromol min(-1) mg(-1) at 50 degrees C. Apparent Michaelis constants at 50 degrees C were Km = 0.65 +/- 0.09 mM for fructose-6-phosphate and Km = 27.8 +/- 4.3 microM for erythrose-4-phosphate. When ribose-5-phosphate replaced erythrose-4-phosphate as an aldose acceptor, Vmax decreased twofold, whereas the Km was 150-fold higher. The molecular mass of the active enzyme is 271 +/- 27 kDa as estimated by gel filtration, whereas the predicted monomer size is 23.96 kDa, suggesting that the native form of the protein is probably a decamer. A readily available source of thermophilic pentose phosphate pathway enzymes including transaldolase may have direct application in enzymatic biohydrogen production.     

Galactose-1-phosphate uridylyltransferase: isolation and properties of a uridylyl-enzyme intermediate.

Galactose-1-P uridylyltransferase catalyzes the interconversion of UDP-galactose and galactose-1-P with UDP-galactose and glucose-1-P by a double displacement pathway involving a uridylyl-enzyme intermediate. The amount of radioactivity incorporated into the protein by uracil-labeled UDP-glucose is decreased by the presence of UDP-galactose, which completes with UDP-glucose for uridylylating the enzyme. The amount of glucose-1-P released upon reaction of the enzyme with UDP-glucose indicates that the dimeric enzyme contains more than one active site per molecule, 1.7 on the average for the most active preparation obtained. This suggests that there is one uridylylation site per subunit and that the subunits are similar or identical. The ureidylyl-enzyme is stable to mild alkaline conditions, 0.10 M NaOH at 60 degrees C for 1 h, but is very sensitive to acid, being largely hydrolyzed after 12 h at pH 3.5 and 4 degrees C. The principal radioactive product resulting from hydrolysis of [uracil-2-14C]uridylyl-ens of the uridylyl-enzyme under the latter conditions is [l]ump. The hydrolytic properties of the uridylyl-enzyme show that the uridylyl moiety is bonded to the protein through a phosphoramidate linkage. Complementary studies on the effects of group selective reagents on the activity of the enzyme suggest that the active site nucleophile to which the uridylyl group is bonded may be a histidine residue. The enzyme is rapidly inactivated by diethyl pyrocarbonate at pH 6 and 0 degrees C and reactivated by NH2OH. UDP-glucose at 0.5 mM fully protects the enzyme against diethyl pyrocarbonate while 70 mM galactose-1-P has only a slight protective effect. Uridylyl-enzyme in inactivated by diethyl pyrocarbonate at no more than 2% of the rate for free enzyme. The enzyme is not inactivated by NaBH4 or by NaBH4 in the presence of UDP-glucose. It is not inhibited by 1 mM pyridoxal phosphate or by 0.5 mM 5-nitrosalicylaldehyde at pH 8.6 and it is not inactivated by NaBH4 in the presence of pyridoxal phosphate. The enzyme is inactivated by 5 to 50 muM p-hydroxymercuribenzoate at pH 8.5, but substrates exert no detectable protective effect against this reagent. It is concluded that the enzyme contains at least one essential sulfhydryl group which is not located in the active site in such a way as to be shielded by substrates.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Characterization of recombinant Aspergillus fumigatus mannitol-1-phosphate 5-dehydrogenase and its application for the stereoselective synthesis of protio and deuterio forms of D-mannitol 1-phosphate

A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpressed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a approximately 40-kDa monomer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of d-mannitol 1-phosphate and d-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 degrees C. NADP(H) showed a marginal activity. Hydrogen transfer from formate to d-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of d-mannitol 1-phosphate or, by applying an analogous procedure using deuterio formate, the 5-[2H] derivative thereof. Following the precipitation of d-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%, based on 200 mM of d-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of d-5-[2H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD+, producing (4S)-[2H]-NADH. Comparison of maximum initial rates for NAD+-dependent oxidation of protio and deuterio forms of D-mannitol 1-phosphate at pH 7.1 and 25 degrees C revealed a primary kinetic isotope effect of 2.9+/-0.2, suggesting that the hydride transfer was strongly rate-determining for the overall enzymatic reaction under these conditions.     

Purification and kinetics of sheep kidney cortex glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase (G-6-PD) is one of the important enzymes, which is responsible for the production of NADPH and ribose-5-phosphate. NADPH is used for the biosynthetic reactions and protection of the cells from free radicals. We have investigated some properties and kinetic mechanism of the sheep kidney cortex G-6-PD. This enzyme has been purified 1,384-fold with a yield of 16.96% and had a specific activity of 27.69 U/mg protein. The purification procedure consists of 2', 5'-ADP-Sepharose 4B affinity chromatography after ultracentrifugation. The sheep kidney cortex G-6-PD was found to operate according to a Ping Pong Bi Bi mechanism. The kinetic parameters from sheep K(m) values for G-6-P and NADP(+) and V(m) were determined to be 0.041+/-0.0043 mM, 0.0147+/-0.001 mM and 28.23+/-0.86 microMol min(-1) mg protein(-1), respectively. The pH optimum was 7.4 and the optimum temperature was 45 degrees C. In our previous study we have found that lamb kidney cortex G-6-PD enzyme obeys 'Ordered Bi Bi' mechanism. We suggest that kinetic mechanism altered due to the aging since sheep G-6-PD uses a 'ping pong' mechanism.     

14C labelling of octulose bisphosphates by L-type pentose pathway reactions in liver in situ and in vitro

The complete reaction sequence of the pentose pathway in vitro was studied by incubating [1-14C] ribose 5-phosphate with rat liver enzyme preparation and assessed by both the rate and extent of formation of the glucose 6-P product. The reactions formed, as intermediates, the 1,8-bisphosphates of D-glycero D-ido octulose (D-g D-i Oct) and D-glycero D-altro octulose, both heavily labelled at C-4 with 14C isotope during the 12h incubation. The formation of the octulose phosphates and the specificity of their isotopic labelling confirms an important prediction of, and contribution by reactions of the L-type pentose phosphate pathway (L-PP) in liver in vitro. Infusion in situ of [6-14C] glucose into the liver of the anaesthetized rabbit resulted in the formation of high specific activity [8-14C] D-g D-i Oct 1,8-P2. The specificity of labelling indicates that the octulose intermediate is formed according to the options of the L-PP mechanism of glucose metabolism in intact liver.     

腾冲嗜热厌氧菌6-磷酸果糖激酶的克隆、表达及其生物活性

6_磷酸果糖激酶(PFK)是糖酵解途径一个关键酶。基于腾冲嗜热厌氧菌基因组中的注释,基因TTE1816可能是PFK的一种,但是,它是否确有生物活性还必须有实验数据的支持。腾冲嗜热厌氧菌在最适温度培养后,提取细菌全蛋白,并采用双向电泳将可溶性蛋白质分离,然后运用质谱鉴定若干染色斑点。实验表明,TTE1816在高温条件下能够表达蛋白质。将TTE1816基因体外克隆至细菌表达载体,并在BL_21大肠杆菌中表达为可溶性蛋白。酶动力学实验表明,重组蛋白TTE1816具有PFK的催化活性,最适反应温度在60℃。它还能够催化葡萄糖、果糖、甘露糖和6_磷酸葡萄糖的磷酸化反应。另外,在高底物浓度和酶浓度的条件下,TTE1816还表现果糖二磷酸酶的特性。结果证明,TTE1816是腾冲嗜热厌氧菌中PFK家族的一个新成员。     

Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7

L-rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms. Glucose-1-phosphate thymidylyltransferase (RmlA, EC 2.7.7.24) catalyzes the first reaction of the four-step pathway of L-rhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate. Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain 7. In this study, we report the heterologous expression of the largest homologue (a 401 residue-long ST0452 protein) and characterization of its thermostable activity. RmlA enzymatic activity of this protein was detected from 65 to 100 degrees C, with a half-life of 60 min at 95 degrees C and 180 min at 80 degrees C. Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein. Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing alpha-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate but not alpha-D-glucosamine-1-phosphate. However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP. Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities. This is the first report of a thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities.     

The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.     

New reaction sequences for the non-oxidative pentose phosphate pathway.

1. Reactions leading to the formation of 14C-labelled volatile compounds and compounds volatile under acid conditions were investigated in a system actively synthesizing hexose 6-phosphates from [U-14C]ribose 5-phosphate by reactions catalysed by enzymes prepared from acetone-dried powder of rat liver; no reactions involving 14C-labelled volatile compounds were detected. Similarly the fixation of 14C-labelled volatile compounds into hexose 6-phosphate could not be detected. 2. A complete carbon balance was made for the reactants, intermediates and products of the reactions involved in the conversion of ribose 5-phosphate into hexose 6-phosphate by enzymes of rat liver. Five additional intermediates of pentose 5-phosphate metabolism in liver were detected, namely D-manno-heptulose 7-phosphate, D-altro-heptulose 1,7-bisphosphate, D-glycero-D-ido-octulose 1,8-bisphosphate, D-glycero-D-altro-octulose 1,8-bisphosphate and D-arabinose 5-phosphate. 3. D-Arabinose 5-phosphate was found to be utilized by a rat liver enzyme preparation to produce both hexose 6-phosphate and triose phosphate. 4. D-Arabinose 5-phosphate was reversibly converted into other pentose 5-phosphates. Paper chromatographic and enzymic evidence indicated that the conversion involved an enzyme tentatively named arabinose phosphate 2-epimerase, which catalyses the following reaction: D-arabinose 5-P in equilibrium D-ribose-5-P. 5. A variety of rat tissues also utilized D-arabinose 5-phosphate to produce both hexose 6-phosphate and triose phosphate and at a rate comparable with that obtained with D-ribose 5-phosphate. 6. A new reaction sequence for the non-oxidative pentose phosphate pathway in liver is proposed.     

Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase

A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.     

Separation and characteristics of galactose-1-phosphate and glucose-1-phosphate uridyltransferase from fruit peduncles of cucumber

Galactose-1-phosphate uridyltransferase (EC 2.7.7.10), responsible for the conversion of galactose-1-phosphate (Gal-1-P) to uridine diphosphate galactose (UDPgal) was examined in fruit peduncles of Cucumis sativus L. Two uridyltransferases (pyrophosphorylases), from I and II, were partially purified and resolved on a diethylamino-ethyl-cellulose column. Form I can utilize glucose-1-phosphate (Glc-1-P), while form II can utilize either Gal-1-P or Glc-1-P, with a preference for Gal-1-P. Form I was more heat stable than form II. Both Glc-1-P and Gal-1-P activities of form II were inactivated at the same rate by heating. The finding of a uridyltransferase with preference for Gal-1-P indicates that cucumber may have a Gal-1-P uridyltransferase (pyrophosphorylase) pathway for the catabolism of stachyose in the peduncles. The absence of the enzyme UDP-glucose-hexose-1-phosphate uridyltransferase (EC 2.7.7.12) in this tissue rules out catabolism by the classical Leloir pathway. The incorporation of carbon from UDPglc into Glc-1-P as opposed to sucrose may be regulated by the activities of the uridyltransferases. Pyrophosphate, in the same concentration range, inhibits UDP-gal formation (K_i=0.58±0.10 mM) and stimulates Glc-1-P formation. The ratio of units of pyrophosphatase to units of Gal-1-P uridyltransferase was higher in peduncles from growing fruit than from unpollinated fruit. Modulation of carbon partitioning through a uridyltransferase pathway may be a factor controlling growth of the cucumber fruit.Abbreviations Gal-1-P Galactose-1-phosphate - Glc-1-P glucose-1-phosphate - UDPgal uridine diphosphate galactose - UDPglc uridine diphosphate glucose Paper No. 6908 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned