Preliminary crystallographic study of an L-asparaginase from Vibrio succinogenes

Crystals of an L-asparaginase from Vibrio succinogenes were obtained with the hanging drop method from ammonium sulphate-containing solutions. The crystals belong to the orthorhombic space group P22(1)2(1) with unit cell dimensions of a = 71.3 A, b = 85.8 A, c = 114.0 A, and contain two tetrameric enzyme molecules per unit cell. There are two subunits in the asymmetric unit; a molecular dyad is coincident with the crystallographic dyad. The crystal lattice is similar to that reported for an Escherichia coli asparaginase. Rotation function calculations have revealed that the V. succinogenes enzyme has 222 point group symmetry in the crystal. The second and third molecular dyads differ, however, from the corresponding E. coli asparaginase dyads by approximately 40 degrees. The crystals diffract to at least 2.2 A resolution and are suitable for X-ray crystallographic structure determination.     

Asparaginase production by human clinical isolates of Vibrio succinogenes

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.     

Asparaginase production by human clinical isolates of Vibrio succinogenes.

Three human isolates of Vibrio succinogenes produced asparaginase. Apparent Km's were 87,220, and 320 microM. The rate of glutamine hydrolysis was between 2.8 and 3.5% of the rate of asparagine hydrolysis. Asparaginase production was not induced by ammonium ions, and enzyme yields were lower than those obtained with the rumen strain.     

固定化细胞生产L—苹果酸动力学及两种酶反应器的比较

研究了产氨短杆菌ＭＡ－２,黄色短杆菌ＭＡ－３的固定化细胞在富马酸铵转化体系中生成Ｌ－苹果酸的动力学参数,同时比较了固定化细胞在填充床及连续机械搅拌反应器中酶转化反应的差异。研究结果表明：当转化率小于４０％时,酶反应在两种反应器所需的停留时间相当。随着转化率的提高,填充床反应器较连续机械搅拌反应器所需的停留时间短且不会因剪切力使固定化颗粒受到损伤,因此,在富马酸铵体系中用固定化酶生产Ｌ－苹果酸采用填     

L-Asparaginase activity in Leptosphaeria michotii. Isolation and properties of two forms of the enzyme

The properties of L-asparaginase (EC 3.5.1.1) in Leptosphaeria michotii (West) Sacc., which has previously been shown to have an activity rhythm, were analyzed. Two forms of L-asparaginase were isolated from acetic acid and ammonium sulfate fractionations followed by DEAE-Sephacel chromatography. The activity of L-asparaginase changed rhythmically with the same period as that of crude extracts, but the rhythms of the two enzyme forms were out of phase. The two asparaginase forms differed in their isoelectric points and the substrate concentrations for attaining half-maximal velocity; non-Michaelis-Menten kinetics for hydrolysis of L-asparagine were observed. Analyses of asparaginase form II by polyacrylamide gel electrophoresis showed that four proteins, irrespective of the phase of the activity rhythm at which the enzyme was extracted, could be detected: asparaginase oligomer (M_r 130 000 to 140 000), its dimer, an aggregate (M_r 500 000 to 600 000) having a low asparaginase activity, and a protein (M_r 60 000) without asparaginase activity; the same proteins were found in asparaginase form I. These results indicate that L. michotii asparaginase could be implicated in a protein complex.     

Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membrane-driven fumarate reduction and energy conservation

Neutral red (NR) functioned as an electronophore or electron channel enabling either cells or membranes purified from Actinobacillus succinogenes to drive electron transfer and proton translocation by coupling fumarate reduction to succinate production. Electrically reduced NR, unlike methyl or benzyl viologen, bound to cell membranes, was not toxic, and chemically reduced NAD. The cell membrane of A. succinogenes contained high levels of benzyl viologen-linked hydrogenase (12.2 U), fumarate reductase (13.1 U), and diaphorase (109.7 U) activities. Fumarate reductase (24.5 U) displayed the highest activity with NR as the electron carrier, whereas hydrogenase (1.1 U) and diaphorase (0.8 U) did not. Proton translocation by whole cells was dependent on either electrically reduced NR or H2 as the electron donor and on the fumarate concentration. During the growth of Actinobacillus on glucose plus electrically reduced NR in an electrochemical bioreactor system versus on glucose alone, electrically reduced NR enhanced glucose consumption, growth, and succinate production by about 20% while it decreased acetate production by about 50%. The rate of fumarate reduction to succinate by purified membranes was twofold higher with electrically reduced NR than with hydrogen as the electron donor. The addition of 2-(n-heptyl)-4-hydroxyquinoline N-oxide to whole cells or purified membranes inhibited succinate production from H2 plus fumarate but not from electrically reduced NR plus fumarate. Thus, NR appears to replace the function of menaquinone in the fumarate reductase complex, and it enables A. succinogenes to utilize electricity as a significant source of metabolic reducing power.     

Extracellular expression and single step purification of recombinant Escherichia coli L-asparaginase II

L-Asparaginase (isozyme II) from Escherichia coli is an important therapeutic enzyme used in the treatment of leukemia. Extracellular expression of recombinant asparaginase was obtained by fusing the gene coding for asparaginase to an efficient pelB leader sequence and an N-terminal 6x histidine tag cloned under the T7lac promoter. Media composition and the induction strategy had a major influence on the specificity and efficiency of secretion of recombinant asparaginase. Induction of the cells with 0.1 mM IPTG at late log phase of growth in TB media resulted in fourfold higher extracellular activity in comparison to growing the cells in LB media followed by induction during the mid log phase. Using an optimized expression strategy a yield of 20,950 UI/L of recombinant asparaginase was obtained from the extracellular medium. The recombinant protein was purified from the culture supernatant in a single step using Ni-NTA affinity chromatography which gave an overall yield of 95 mg/L of purified protein, with a recovery of 86%. This is approximately 8-fold higher to the previously reported data in literature. The fluorescence spectra, analytical size exclusion chromatography, and the specific activity of the purified protein were observed to be similar to the native protein which demonstrated that the protein had folded properly and was present in its active tetramer form in the culture supernatant.     

Derepression of asparaginase II during exponential growth of Saccharomyces cerevisiae on ammonium ion

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is sensitive to nitrogen catabolite repression. In cell cultures growing in complete ammonia medium, asparaginase II synthesis is repressed in the early exponential phase but becomes derepressed in the midexponential phase. When amino acids such as glutamine or asparagine replace ammonium ion in the growth medium, the enzyme remains repressed into the late exponential phase. The three nitrogen compounds permit a similar rate of cell growth and are assimilated at nearly the same rate. In the early exponential phase the internal amino acid pool is larger in cells growing with glutamine or asparagine than in cells growing with ammonium sulfate as the sole source of nitrogen.     

A third crystal form of Wolinella succinogenes quinol:fumarate reductase reveals domain closure at the site of fumarate reduction.

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD-binding domain and the capping domain [Lancaster, C.R.D., Kr?ger, A., Auer, M., & Michel, H. (1999) Nature 402, 377--385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 A resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14 degrees relative to the FAD-binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane-bound and soluble fumarate reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site-directed mutagenesis strongly support a common mechanism for fumarate reduction in this superfamily of enzymes.     

Cloning and expression of the genes of two fumarate reductase subunits from Wolinella succinogenes

The fumarate reductase complex of the anaerobic bacterium Wolinella succinogenes catalyzes the electron transfer from menaquinol to fumarate. Two structural genes coding for subunits of the enzyme have been cloned in Escherichia coli. The genes were isolated from a lambda EMBL3 phage gene bank by immunological screening and subcloned in an expression vector. The genes frdA and frdB, which encode the FAD protein (Frd A, Mr 79,000) and the iron-sulfur protein (Frd B, Mr 31,000) of the fumarate reductase complex, were cloned together with a W. succinogenes promoter. The gene order was promoter-frdA-frdB. The FAD protein and the iron-sulfur protein were expressed in the correct molar mass in E. coli from the clones. The identity of the frdA gene and the suggested polarity were confirmed by comparing the amino-terminal sequence of the Frd A protein with that predicted from the 5'-terminal nucleotide sequence of frdA. The frdA and frdB genes are present only once in the genome. A region downstream of frdB, possibly a gene encoding cytochrome b of the fumarate reductase complex, hybridizes with a second site in the genome.     

Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni

The epsilon-proteobacteria Helicobacter pylori and Campylobacter jejuni are both human pathogens. They colonize mucosal surfaces causing severe diseases. The membrane protein complex QFR (quinol:fumarate reductase) from H. pylori has previously been established as a potential drug target, and the same is likely for the QFR from C. jejuni. In the present paper, we describe the cloning of the QFR operons from the two pathogenic bacteria H. pylori and C. jejuni and their expression in Wolinella succinogenes, a non-pathogenic -proteobacterium. To our knowledge, this is the first documentation of heterologous membrane protein production in W. succinogenes. We demonstrate that the replacement of the homologous enzyme from W. succinogenes with the heterologous enzymes yields mutants where fumarate respiration is fully functional. We have isolated and characterized the heterologous QFR enzymes. The high quality of the enzyme preparation enabled us to determine unequivocally by analytical ultracentrifugation the homodimeric state of the three detergent-solubilized heterotrimeric QFR enzymes, to accurately determine the different oxidation-reduction ('redox') midpoint potentials of the six prosthetic groups, the Michaelis constants for the quinol substrate, maximal enzymatic activities and the characterization of three different anti-helminths previously suggested to be inhibitors of the QFR enzymes from H. pylori and C. jejuni. This characterization allows, for the first time, a detailed comparison of the QFR enzymes from C. jejuni and H. pylori with that of W. succinogenes.     

Regulation of asparaginase, glutamine synthetase, and glutamate dehydrogenase in response to medium nitrogen concentrations in a euryhaline chlamydomonas species

The ammonium assimilatory enzymes glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.3) were investigated for a possible role in the regulation of asparaginase (EC 3.5.1.1) in a Chlamydomonas species isolated from a marine environment. Cells grown under nitrogen limitation (0.1 millimolar NH(4) (+), NO(3) (-), or l-asparagine) possessed 6 times the asparaginase activity and approximately one-half the protein of cells grown at high nitrogen levels (1.5 to 2.5 millimolar). Biosynthetic glutamine synthetase activity was 1.5 to 1.8 times greater in nitrogen-limited cells than cells grown at high levels of the three nitrogen sources.Conversely, glutamate dehydrogenase (both NADH- and NADPH-dependent activities) was greatest in cells grown at high levels of asparagine or ammonium, while nitrate-grown cells possessed little activity at all concentrations employed. For all three nitrogen sources, glutamate dehydrogenase activity was correlated to the residual ammonium concentration of the media after growth (r = 0.88 and 0.94 for NADH- and NADPH-dependent activities, respectively).These results suggest that glutamate dehydrogenase is regulated in response to ambient ammonium levels via a mechanism distinct from asparaginase or glutamine synthetase. Glutamine synthetase and asparaginase, apparently repressed by high levels of all three nitrogen sources, are perhaps regulated by a common mechanism responding to intracellular nitrogen depletion, as evidenced by low cellular protein content.     

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes.

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.     

Unsaturated organic acids as terminal electron acceptors for reductase chains of anaerobic bacteria

This paper summarizes the current knowledge of unsaturated organic acids in their role as terminal electron acceptors of anaerobic bacteria. The mechanisms and enzyme systems involved in the reduction of fumarate by Escherichia coli, Wolinella succinogenes, and some species of the genus Shewanella are considered. Particular attention is given to reduction of the double bond of the unnatural compound methacrylate by the sigma-proteobacterium Geobacter sulfurreducens Am-1. Soluble periplasmic flavocytochromes c, found in bacteria of the genera Shewanella and Geobacter, are involved in the hydration of fumarate (in Shewanella species) and methacrylate (in G. sulfurreducens Am-1). In E. coli and W. succinogenes, fumarate is reduced in cytosol by membrane-bound fumarate reductases. The prospects for research into organic acid reduction at double bonds in bacteria are discussed.     

Studies on the relatedness of bacterial fumarate reductases

Abstract A collection of 10 Gram-negative bacteria was examined for the presence of a fumarate reductase related to that of Escherichia coli K-12. When the frd genes encoding the E. coli enzyme were used as DNA:DNA hybridization probes good signals were obtained from all members of the family Enterobacteriaceae. No significant hybridization was detected, even under non-stringent conditions, with the well characterized fumarate reducer Vibrio succinogenes or with Pseudomonas aeruginosa . These findings were confirmed and extended by immuno-diffusion studies using cell membranes and antiserum against the E. coli reductase. Precipitin lines were observed in all cases where frd homologies were detected. It was concluded that the V. succinogenes enzyme differs extensively from the E. coli fumarate reductase.     

Periplasmic methacrylate reductase activity in Wolinella succinogenes

The cell homogenate and the soluble cell fraction of Wolinella succinogenes grown with formate and fumarate catalyzed the oxidation of benzyl viologen radical by methacrylate [apparent Km=0.23 mM, Vmax=1.0 U (mg cell protein) -1] or acrylate [apparent Km=0.50 mM, Vmax=0.77 U (mg cell protein) -1]. Crotonate did not serve as an oxidant. A mutant of W. succinogenes lacking the fccABC operon was unable to catalyze methacrylate or acrylate reduction. In contrast, the inactivation of fccC alone had no effect on these activities. Methacrylate reduction by benzyl viologen radical was not catalyzed by fumarate reductase isolated from the membrane of W. succinogenes. Cells grown with formate and fumarate did not catalyze methacrylate reduction by formate, and W. succinogenes did not grow with formate and methacrylate as catabolic substrates. The results suggest that the reduction of methacrylate or acrylate by benzyl viologen radical is most likely catalyzed either by the periplasmic flavoprotein FccA or by a complex consisting of FccA and the predicted c-type cytochrome FccB. The metabolic function of the fccABC operon remains unknown.     

Improved Growth Media for Vibrio succinogenes

Five new culture media for Vibrio succinogenes are described. Of these, a medium composed of 0.4% yeast extract, 100 mM ammonium formate, 120 mM sodium fumarate, and 0.05% sodium thioglycolate, pH 7.3, supports the best growth.     

Construction of expression systems for Escherichia coli asparaginase II and two-step purification of the recombinant enzyme from periplasmic extracts.

Isoenzyme II of Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) is among the few enzymes of major therapeutic importance, being used in the treatment of acute lymphoblastic leukemia. We have constructed several inducible expression systems that overproduce asparaginase II from recombinant plasmids. The most efficient of these systems consists of plasmid pTWE1, a derivative of pT7-7, and an ansB- strain of E. coli, CU1783. These cells produce and secrete amounts of asparaginase II that account for 10-15% of the total cellular protein. Most of the active recombinant enzyme can be released from the periplasmic space by a simple osmotic shock procedure. From the resulting material homogeneous asparaginase II was obtained by a two-step procedure. Overall yields of purified asparaginase were 10-15 mg asparaginase II per liter of E. coli culture. The recombinant enzyme appeared identical to conventionally purified preparations.     

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli.

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, predent in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Stephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.     

Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase

L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.