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1.
Glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes the transfer of the C2 amino group of glutamate 1-semialdehyde (GSA) to the C1 position. Nucleic acid sequences encoding this enzyme from wild type and a gabaculine (GAB) resistant strain of Synechococcus have been cloned and overexpressed in Escherichia coli. Tolerance to GAB of the mutant GSA-AT resulted from a point mutation, Met-248-Ile, in the middle of the polypeptide chain accompanied by a deletion of three amino acids close to the NH2 terminus but can also be effected by the point mutation alone. Purified enzymes from these two strains contain vitamin B6 and use a typical ping-pong Bi-Bi mechanism, in which 4,5-diaminovalerate (DAVA) is a likely intermediate. The catalytic efficiency (Kcat/Km) of wild-type GSA-AT for GSA is about 3 times larger than that of the mutant enzyme. Comparison of substrate specificities (kmax/Km) for GSA and various analogues reveals that wild-type GSA-AT has values that are about 2-20 times larger than those of the mutant enzyme, except in the case of GAB for which the specificity is 2-3 orders of magnitude larger. These differences are attributed to impaired prototropic rearrangement and transaldimination by mutant GSA-AT. They lead to accumulation of quinonoid and other intermediates upon addition of various substrates such as ALA and DOVA, as well as to instability of their aldimines (418 nm) upon Sephadex gel filtration. 相似文献
2.
Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C5 pathway for the synthesis of δ-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5?μM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100?μM gabaculine, this enzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemL GR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemL WT had retained its sensitivity. Southern hybridisation using gene probes for hemL, amp r and cm r confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10?μM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine. 相似文献
3.
Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the
C5 pathway for the synthesis of δ-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor
of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5 μM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100 μM gabaculine, this enzyme has undergone two changes in structure:
a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To
establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination,
of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemL
GR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while
a wild-type recombinant produced using pHS7-hemL
WT had retained its sensitivity. Southern hybridisation using gene probes for hemL, amp
r
and cm
r
confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll
accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10 μM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is
dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide
vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine.
Received: 7 October 1996 / Accepted: 15 January 1997 相似文献
4.
Cyanobacterial GR6 glutamate-1-semialdehyde aminotransferase: a novel enzyme-based selectable marker for plant transformation 总被引:2,自引:0,他引:2
Angelica Giancaspro Daniele Rosellini Antonio Blanco Agata Gadaleta 《Plant cell reports》2001,20(4):296-300
The mutant glutamate-1-semialdehyde aminotransferase (GSA-AT) enzyme encoded by the hemL gene of the gabaculine-resistant cyanobacterium Synechococcus PCC6301 strain GR6 was expressed in tobacco following Agrobacterium-mediated transformation of leaf discs. When targeted to plastids, the GR6 hemL gene product conveyed gabaculine resistance to transgenic plants. Selection using 50 and 100 µM gabaculine was shown to produce two distinct explant phenotypes: 'greens' and 'whites'. T1 progeny displayed Mendelian segregation ratios, and PCR analysis demonstrated the 'green' phenotype corresponded with the presence of the GR6 hemL gene. Furthermore, 'whites' could be rescued after 9 days growth on solid media containing between 5 µM and 25 µM gabaculine, allowing the potential use of this system for the isolation of gabaculine-sensitive transformants in mutagenesis screening. The use of GR6 hemL as a selectable marker gene provides a novel enzyme-based method for the selection of transgenic regenerants without the need for antibiotic-resistance markers. 相似文献
5.
Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key regulatory enzyme, which converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA) in chlorophyll biosynthesis. ALA is the universal precursor for the synthesis of chlorophyll, heme, and other tetrapyrroles. To study the regulation of chlorophyll biosynthesis in Brassica napus, two cDNA clones of GSA-AT were isolated for genetic manipulation. A SalI-XbaI fragment from one of the two cDNA clones of GSA-AT was used for recombinant protein expression by inserting it at the 3' end of a calmodulin-binding-peptide (CBP) tag of the pCaln vector. The CBP tagged recombinant protein, expressed in Escherichia coli, was purified to apparent homogeneity in a one step purification process using a calmodulin affinity column. The purified CBP tagged GSA-AT is biologically active and has a specific activity of 16.6 nmol/min/mg. Cleavage of the CBP tag from the recombinant protein with thrombin resulted in 9.2% loss of specific activity. However, removal of the cleaved CBP tag from the recombinant protein solution resulted in 60% loss of specific activity, suggesting possible interactions between the recombinant protein and the CBP tag. The enzyme activity of the CBP tagless recombinant protein, referred as TR-GSA-AT hereafter, was not affected by the addition of pyridoxamine 5' phosphate (PMP). Addition of glutamate and pyridoxal 5' phosphate (PLP) to the TR-GSA-AT enhanced the enzyme activity by 3-fold and 3.6-fold, respectively. Addition of both glutamate and PLP increased the enzyme activity by 4.6-fold. Similar to the GSA-AT of B. napus, the active TR-GSA-AT is a dimeric protein of 88 kDa with 45.5 kDa subunits. As the SalI-XbaI fragment encodes a biologically active GSA-AT that has the same molecular mass as the native GSA-AT, it is concluded that the SalI-XbaI fragment is the coding sequence of GSA-AT. The highly active polyclonal antibodies generated from TR-GSA-AT were used for the detection of GSA-AT of B. napus. 相似文献
6.
Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco 总被引:2,自引:0,他引:2
Anna Zavgorodnyaya Jutta Papenbrock Bernhard Grimm 《The Plant journal : for cell and molecular biology》1997,12(1):169-178
Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an amino-terminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and, consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway. 相似文献
7.
Glutamate-l-semialdehyde (GSA) aminotransferase catalyses the final step in the C5 pathway converting glutamate to the tetrapyrrole precursor δ-aminolaevulinic acid. This enzyme is sensitive to gabaculine (2,3-dihydro-3-amino benzoic acid) and to 4-amino-5-fluoropentanoic acid (AFPA), which are irreversible, mechanism-based inhibitors of pyridoxal phosphatedependent enzymes. Spontaneous mutants of Synechococcus PCC6301 resistant to these inhibitors contain altered enzyme that displays corresponding resistance to high concentrations of the inhibitor. The enzyme from strain GR6, resistant to both inhibitors, contains a three-amino-acid deletion at positions 5–7 and a Met248 → Ile substitution. The enzyme from strain K40 resistant to AFPA but not to gabaculine, contains a Ser163 → Thr substitution. GSA aminotransferases containing either the deletion or the substitution that are characteristic of the GR6 mutant were produced in Escherichia coli using the expression vector pMalc2. These engineered mutant enzymes were characterized in terms of their catalytic parameters and sensitivities to gabaculine and AFPA. Furthermore, maltose binding protein/aminotransferase fusion proteins were characterized spectrophotometrically to monitor the interaction of bound cofactor with diamino- and dioxocompounds related to the substrate and both inhibitors. Results were compared with those for similarly produced recombinant wild-type, K40 and GR6 GSA aminotransferases. The engineered products with either the N-terminal deletion or the Met248 → Ile substitution displayed catalytic efficiencies that were intermediate between the wild-type and GR6 or K40 enzymes. However, with respect to their absorption spectra, sensitivity to inhibitors and the reactivity of bound cofactor, they were essentially wild-type. These in vitro studies demonstrate that both changes in enzyme structure are necessary to obtain the distinctive properties of the GR6 aminotransferase, including resistance to high concentrations of gabaculine and AFPA. 相似文献
8.
Rosellini D Capomaccio S Ferradini N Savo Sardaro ML Nicolia A Veronesi F 《Plant cell reports》2007,26(7):1035-1044
A selectable marker gene (SMG), usually conferring resistance to an antibiotic or herbicide, is generally introduced into
the plant cells with the gene(s) for the trait of interest to allow only the cells that have integrated and express the foreign
sequences to regenerate into a plant. The availability of several SMGs for each plant species is useful for both basic and
applied research to combine several genes of interest in the same plant. A selection system based on gabaculine (3-amino-2,3-dihydrobenzoic
acid) as the selective substance and the bacterial hemL gene [encoding a mutant for of the enzyme glutamate 1-semialdehyde aminotransferase (GSA-AT)] as the SMG was previously
used for genetic transformation of tobacco. The hemL gene is a good candidate for a safe SMG, because GSA-AT is present in all plants and is likely involved in one metabolic
step only, so that unintended effects of its overexpression in plants are not probable. In this work, we have compared this
new selection system with the conventional, kanamycin-based system for alfalfa Agrobacterium-mediated transformation. The hemL and NptII genes were placed together into a T-DNA under the control of identical promoters and terminators. We show that the gabaculine-based
system is more efficient than the conventional, kanamycin-based system. The inheritance of hemL was Mendelian, and no obvious phenotypic effect of its expression was observed. 相似文献
9.
Site-directed mutagenesis of Petunia hybrida 5-enolpyruvylshikimate-3-phosphate synthase: Lys-23 is essential for substrate binding 总被引:1,自引:0,他引:1
Q K Huynh S C Bauer G S Bild G M Kishore J R Borgmeyer 《The Journal of biological chemistry》1988,263(24):11636-11639
Chemical modification of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine), with pyridoxal 5'-phosphate suggested that Lys-22 (equivalent to Lys-23 of the Petunia hybrida enzyme) is a potential active site residue (Huynh, Q. K., Kishore, G. M., and Bild, G. S. (1988) J. Biol. Chem. 263, 735-739). To investigate the possible role of this residue in the reaction mechanism, we have used site-directed mutagenesis to replace Lys-23 of the P. hybrida enzyme with 3 other amino acid residues: Ala, Glu, and Arg. Analysis of these mutant enzymes indicates that of these only the Lys-23 to Arg mutant enzyme is active; the other two replacements (Ala and Glu) result in inactivation of the enzyme. Two of the mutant enzymes (Lys-23 to Arg and Ala) were purified to homogeneity and characterized. The purified Lys-23 to Arg mutant enzyme is less sensitive than the wild type enzyme to pyridoxal 5'-phosphate. It showed identical Km values for substrates and a 5-fold higher I50 value for glyphosate in comparison with those from the wild type enzyme. Binding studies using fluorescence measurements revealed that the substrate shikimate 3-phosphate and glyphosate were able to bind the purified Lys-23 to Arg mutant enzyme but not to the purified catalytically inactive Lys-23 to Ala mutant enzyme. The above results suggest that the cationic group at position 23 of the enzyme may play an important role in substrate binding. 相似文献
10.
Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli 总被引:1,自引:0,他引:1
L J Kimlova C Pyne K Keshavjee J Huy G Beebakhee A L Bognar 《Archives of biochemistry and biophysics》1991,284(1):9-16
The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme. 相似文献
11.
Enoyl-[acyl-carrier-protein] (ACP) reductase is a key enzyme in type II fatty-acid synthases that catalyzes the last step in each elongation cycle. The FabI component of Bacillus subtilis (bsFabI) was identified in the genomic data base by homology to the Escherichia coli protein. bsFabI was cloned and purified and exhibited properties similar to those of E. coli FabI, including a marked preference for NADH over NADPH as a cofactor. Overexpression of the B. subtilis fabI gene complemented the temperature-sensitive growth phenotype of an E. coli fabI mutant. Triclosan was a slow-binding inhibitor of bsFabI and formed a stable bsFabI.NAD(+). triclosan ternary complex. Analysis of the B. subtilis genomic data base revealed a second open reading frame (ygaA) that was predicted to encode a protein with a relatively low overall similarity to FabI, but contained the Tyr-Xaa(6)-Lys enoyl-ACP reductase catalytic architecture. The purified YgaA protein catalyzed the NADPH-dependent reduction of trans-2-enoyl thioesters of both N-acetylcysteamine and ACP. YgaA was reversibly inhibited by triclosan, but did not form the stable ternary complex characteristic of the FabI proteins. Expression of YgaA complemented the fabI(ts) defect in E. coli and conferred complete triclosan resistance. Single knockouts of the ygaA or fabI gene in B. subtilis were viable, but double knockouts were not obtained. The fabI knockout was as sensitive as the wild-type strain to triclosan, whereas the ygaA knockout was 250-fold more sensitive to the drug. YgaA was renamed FabL to denote the discovery of a new family of proteins that carry out the enoyl-ACP reductase step in type II fatty-acid synthases. 相似文献
12.
1. A precursor to small stable RNA, 10Sa RNA, accumulates in large amounts in a temperature sensitive RNase E mutant at non-permissive temperatures, and somewhat in an rnc (RNase III-) mutant, but not in an RNase P- mutant (rnp) or wild type E. coli cells. 2. Since p10Sa RNA was not processed by purified RNase E and III in customary assay conditions, we purified p10Sa RNA processing activity about 700-fold from wild type E. coli cells. 3. Processing of p10Sa RNA by this enzyme shows an absolute requirement for a divalent cation with a strong preference for Mn2+ over Mg2+. Other divalent cations could not replace Mn2+. 4. Monovalent cations (NH+4, Na+, K+) at a concentration of 20 mM stimulated the processing of p10Sa RNA and a temperature of 37 degrees C and pH range of 6.8-8.2 were found to be optimal. 5. The enzyme retained half of its p10Sa RNA processing activity after 30 min incubation at 50 degrees C. 6. Further characterization of this activity indicated that it is RNase III. 7. To further confirm that the p10Sa RNA processing activity is RNase III, we overexpressed the RNase III gene in an E. coli cells that lacks RNase III activity (rnc mutant) and RNase III was purified using one affinity column, agarose.poly(I).poly(C). 8. This RNase III preparation processed p10Sa RNA in a similar way as observed using the p10Sa RNA processing activity purified from wild type E. coli cells, confirming that the first step of p10Sa RNA processing is carried out by RNase III. 相似文献
13.
The Escherichia coli hemL gene encodes glutamate 1-semialdehyde aminotransferase. 总被引:6,自引:3,他引:3
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delta-Aminolevulinic acid (ALA), the first committed precursor of porphyrin biosynthesis, is formed in Escherichia coli by the C5 pathway in a three-step, tRNA-dependent transformation from glutamate. The first two enzymes of this pathway, glutamyl-tRNA synthetase and Glu-tRNA reductase, are known in E. coli (J. Lapointe and D. Söll, J. Biol. Chem. 247:4966-4974, 1972; D. Jahn, U. Michelsen, and D. Söll, J. Biol. Chem. 266:2542-2548, 1991). Here we present the mapping and cloning of the gene for the third enzyme, glutamate 1-semialdehyde (GSA) aminotransferase, and an initial characterization of the purified enzyme. Ethylmethane sulfonate-induced mutants of E. coli AB354 which required ALA for growth were isolated by selection for respiration-defective strains resistant to the aminoglycoside antibiotic kanamycin. Two mutations were mapped to min 4 at a locus named hemL. Map positions and resulting phenotypes suggest that hemL may be identical with the earlier described porphyrin biosynthesis mutation popC. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding clone pLC4-43 of the Clarke-Carbon bank (L. Clarke and J. Carbon, Cell 9:91-99, 1976) allowed the isolation of the gene. Physical mapping showed that hemL mapped clockwise next to fhuB. The hemL gene product was overexpressed and purified to apparent homogeneity. The pure protein efficiently converted GSA to ALA. The reaction was stimulated by the addition of pyridoxal 5' -phosphate or pyridoxamine 5' -phosphate and inhibited by gabaculine or aminooxyacetic acid. The molecular mass of the purified GSA aminotransferase under denaturing conditions was 40,000 Da, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has apparent native molecular mass of approximately 80,000 Da, as determined by rate zonal sedimentation on glycerol gradients and molecular sieving through Superose 12, which indicates a homodimeric alpha2, structure of the protein. 相似文献
14.
Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties. 总被引:3,自引:1,他引:2
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A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3. 相似文献
15.
Incubation of anticapsin with the purified glucosamine synthetase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase, amino transferring, EC 5.3.1.19) from Escherichia coli, Pseudomonas aeruginosa, Arthrobacter aurescens and Bacillus thuringiensis led to the formation of an inactive enzyme irreversibly modified. The inactivation reaction followed pseudo-first-order kinetics. The rate of the inactivation reaction at various concentrations of anticapsin exhibited saturation kinetics, implying that anticapsin binds reversibly to the enzyme prior to inactivation. The determined Kinact is in the range of 10(-5) M (B. thuringiensis) and 10(-6) M (E. coli, P. aeruginosa, A. aurescens ). The addition of glutamine protected the amidotransferase from inactivation by anticapsin . The anticapsin was demonstrated to be a mixed type or competitive inhibitor with respect to glutamine with a Ki value of 10(-6) to 10(-7) M. Reaction of anticapsin with the enzyme exhibits the characteristics of affinity labelling of the glutamine binding site. Chemical modification of the enzyme thiol group with various reagents, 5,5'-dithiobis-(2-nitrobenzoic) acid, 6,6'- dithiodinicotinic acid, 1,1'- dithiodiformamidine , N-ethylmaleimide and iodoacetamide, resulted in an inactive enzyme. 相似文献
16.
Inosine 5''-monophosphate dehydrogenase of Escherichia coli. Purification by affinity chromatography, subunit structure and inhibition by guanosine 5''-monophosphate. 总被引:3,自引:0,他引:3
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Escherichia coli IMP dehydrogenase (EC 1.2.1.14) was purified by affinity chromatography on immobilized nucleotides. The enzyme binds to agarose-bound 8-(6-aminohexyl)-AMP, N6-(6-aminohexyl)-AMP and 8-(8-amino-octyl)-IMP but not to immobilized NAD+ or Cibacron Blue F3G-A. AMP proved to be an effective eluent. A large-scale purification scheme in which 8-(6-aminohexyl)-AMP-agarose was used resulted in a homogeneous preparation of IMP dehydrogenase. The enzyme was also purified by immunoprecipitation with monospecific antisera. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and tryptic 'finger-printing' demonstrated that IMP dehydrogenase comprises identical subunits of mol.wt. 58000. Trypsin and Pronase cleave the 58000-mol.wt. subunit into peptides of mol.wts. 42000 and 14000, with a concomitant decrease in enzyme activity. These observations rationalize much of the contradictory data on the subunit composition of the enzyme found in the literature. GMP appears to be a competitive inhibitor with respect to IMP, with no evidence for regulatory behaviour being found. The two purification procedures were also used to purify inactive mutant enzymes from guaB mutant strains of E. coli. 相似文献
17.
The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18. This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes. The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation. The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase. The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand. The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity. The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis. Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers. The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition. 相似文献
18.
Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,1-aminomutase; EC 5.4.3.8; GSA-AT) catalyzes the transfer of the amino group on carbon 2 of glutamate 1-semialdehyde (GSA) to the neighboring carbon 1 to form delta-aminolevulinic acid (ALA). To gain insight into the mechanism of this enzyme, possible intermediates were tested with purified enzyme and the reaction sequence was followed spectroscopically. While 4,5-dioxovaleric acid (DOVA) was efficiently converted to ALA by the pyridoxamine 5'-phosphate (PMP) form of the enzyme, 4,5-diaminovaleric acid (DAVA) was a substrate for the pyridoxal 5'-phosphate (PLP) form of GSA-AT. Thus, both substances are reaction intermediates. The purified enzyme showed an absorption spectrum with a peak around 338 nm. Addition of PLP led to increased absorption at 338 nm and a new peak around 438 nm. Incubation of the purified enzyme with PMP resulted in an additional absorption peak at 350 nm. The reaction of the PLP and PMP form of the enzyme with GSA allowed the detection of a series of peaks which varied in their intensities in a time-dependent manner. The most drastic changes to the spectrum that were observed during the reaction sequence were at 495 and 540 nm. Some of the detected absorption bands during GSA-AT catalysis were previously described for several other aminotransferases, indicating the relationship of the mechanisms. The reaction of the PMP form of the enzyme with DOVA resulted in a similar spectrum as described above, while the spectrum for the conversion of DAVA by the PLP form of the enzyme indicated a different mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
19.
Robert R. Rando F.W. Bangerter 《Biochemical and biophysical research communications》1977,76(4):1276-1281
The naturally occurring amino acid gabaculine ((?)-5-amino-1,3 cyclohexadiene carboxylic acid) is a potent irreversible inhibitor of mouse brain γ-aminobutyric acid (GABA)-α-ketoglutaric acid transaminase. When administered I.P. gabaculine, irreversibly inhibits the mouse brain enzyme in a time dependent fashion. Concomitant with this inhibition is a rise in endogenous brain GABA levels. Administration of gabaculine at a concentration of 100 mg/kg mouse leads to the complete inhibition of the enzyme after 4 hrs. Brain levels of GABA continually rise after the administration of the drug. After 20 hrs they are 15–20 times higher than levels in the untreated animals. 相似文献
20.
Transposition of Tn5 in Escherichia coli is regulated by two transposon-encoded proteins: transposase (Tnp), promoting transposition preferentially in cis, and the trans-acting inhibitor (Inh). Two separate transposase mutants were isolated that replace glutamate with lysine at position 110 (EK110) and at position 345 (EK345). The EK transposase proteins increase the Tn5 transposition frequency 6- to 16-fold in cis and enhance the ability of transposase to act in trans. The purified mutant transposase proteins interact with transposon outside end DNA differently from the wild-type protein, resulting in the formation of a novel complex in gel retardation assays. During characterization of the transposase proteins in the absence of inhibitor, we found that wild-type transposase itself has a transposition-inhibiting function and that this inhibition is reduced for the mutant proteins. We present a model for the regulation of Tn5 transposition, which proposes the existence of two transposase species, one cis-activating and the other trans-inhibiting. The phenotype of the EK transposase mutants can be explained by a shift in the ratio of these two species. 相似文献