Glutamate 1-semialdehyde Aminotransferase Research Articles

Activity and spectroscopic properties of the Escherichia coli glutamate 1-semialdehyde aminotransferase and the putative active site mutant K265R.

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)

Purification and Characterization of Glutamate 1-Semialdehyde Aminotransferase from Barley Expressed in Escherichia coli

The immediate precursor in the synthesis of tetrapyrroles is Delta-aminolevulinate (ALA). ALA is synthesized from glutamate in higher plants, algae, and certain bacteria. Glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8) (GSA-AT), the third enzyme involved in this metabolic pathway, catalyzes the transamination of GSA to form ALA. The gene encoding this aminotransferase has previously been isolated from barley (Hordeum vulgare) and inserted into an Escherichia coli expression vector. We describe herein the purification of this recombinant barley GSA-AT expressed in Escherichia coli. Coexpression of GroEL and GroES is required for isolation of active aminotransferase from the soluble protein fraction of Escherichia coli. Purified GSA-AT exhibits absorption maxima characteristic of vitamin B(6)-containing enzymes. GSA-AT is primarily in the pyridoxamine form when isolated and can be interconverted between this and the pyridoxal form by addition of 4,5-dioxovalerate and 4,5-diaminovalerate. The conversion of GSA to ALA under steady-state conditions exhibited typical Michaelis-Menten kinetics. Values for K(m) (d,l-GSA) and k(cat) were determined to be 25 micromolar and 0.11 per second, respectively, by nonlinear regression analysis. Stimulation of ALA synthesis by increasing concentrations of d,l-GSA at various fixed concentrations of 4,5-diaminovalerate supports the hypothesis that 4,5-diaminovalerate is the intermediate in the synthesis of ALA.

Components of chlorophyll biosynthesis in a barley albina mutant unable to synthesize ?-aminolevulinic acid by utilizing the transfer RNA for glutamic acid

Components of chlorophyll biosynthesis were investigated in the plastid-ribosome-deficient albostrians mutant of barley (Hordeum vulgare L.). Compared with green leaves, white leaves lacked chloroplast tRNA(Glu) and 16S ribosomal RNA, but contained a much higher level of the mRNA for glutamate 1-semialdehyde aminotransferase. Substantial amounts of protochlorophyllide were accumulated when the mutant was incubated in a solution of δ-aminolevulinic acid. The level of protochlorophyllide oxidoreductase mRNA (PCOR, EC 1.6.99.1.) in etiolated albostrians plants reached only about 50% of the level in wild-type plants. In addition the content of PCOR protein and the activity of chlorophyll synthetase were distinctly lower than in the wild-type. Mutant and wild-type barley seedlings which were grown under a daily light/dark regime and were therefore nonetiolated both possessed PCOR mRNA. The data presented may help explain the albino phenotype of this mutant. The results are discussed in relation to biosynthesis of tetrapyrrols in higher plants, regulation of chlorophyll biosynthesis and the action of a plastidderived signal involved in the expression of certain nuclear genes.

The role of Lys272 in the pyridoxal 5-phosphate active site of Synechococcus glutamate-1-semialdehyde aminotransferase.

Glutamate-1-semialdehyde (GSA) aminotransferase catalyzes transfer of the C2 amino group of glutamate 1-semialdehyde to the C1 position to yield the tetrapyrrole precursor 5-aminolevulinate. Based on spectrophotometric and steady-state data, GSA aminotransferase is a B6-containing enzyme which uses a ping-pong bi-bi mechanism described for other aminotransferases. A putative active-site lysine at position 272 of Synechococcus GSA aminotransferase was replaced by Arg, Ile or Glu, and genes encoding the corresponding three site directed mutants were expressed in Escherichia coli. The catalytic competence of the resulting enzymes was determined. The similarity of the absorbance spectra of pyridoxal-P-treated forms of Lys272----Arg, Lys272----Ile, Lys272----Glu with free pyridoxal-P indicates that enzyme-bound pyridoxal-P does not form an internal aldimine in in these three site-directed mutants. Whereas Lys----Ile and Lys----Glu form only stable ketimines and aldimines with GSA and its analogues, addition of these compounds to the pyridoxamine-P and pyridoxal-P forms of Lys----Arg induces slow spectral changes, indicating the catalysis of a half-reaction with GSA, 4,5-dioxovalerate and 4,5-diaminovalerate. 5-Aminolevulinate apparently binds with both coenzyme forms of Lys272----Arg, however significant tautomeric rearrangement is only observed with the pyridoxal-P form. It is suggested that Lys272 is the covalent pyridoxal-P-binding site and that this catalytically active lysine residue channels the overall transamination reaction towards 5-aminolevulinate. The second-half reaction (4,5-diaminovalerate in equilibrium with 5-aminolevulinate) is possibly supported by the formation of an internal aldimine which correctly positions the C4 amino group of 4,5-diaminovalerate relative to the enzyme-bound pyridoxal-P.

The enantioselective participation of (S)- and (R)-diaminovaleric acids in the formation of δ-aminolevulinic acid in cyanobacteria

Although it is recognized that 4,5-diaminovaleric acid, formed from glutamate 1-semialdehyde, functions as the intermediate in the last step of delta-aminolevulinic acid formation from glutamate, the enantioselectivity of the participating glutamate 1-semialdehyde aminotransferase for 4,5-diaminovaleric acid has remained unknown. In the present work the involvement of (S)- and (R)-4,5-diaminovaleric acids, newly available by organic synthesis, was investigated, using glutamate 1-semialdehyde aminotransferase from Synechococcus. The preferred enantiomer was (S)-4,5-diaminovalerate. In experiments on the transformation of (S)-4,5-diaminovalerate to delta-aminolevulinate it was found that glutamate 1-semialdehyde aminotransferase was unusual among aminotransferases in that the common amino acceptors pyruvate, oxaloacetate, alpha-ketoglutarate were inactive, while 4,5-dioxovaleric acid could be utilized as a sluggish amino acceptor in place of glutamate 1-semialdehyde. In conclusion, glutamate 1-semialdehyde aminotransferase is highly but not absolutely enantioselective for (S)-4,5-diaminovaleric acid, and 4,5-dioxovaleric acid can function as amino acceptor not because of a physiological role in the C5 pathway of delta-aminolevulinic acid formation, but because of its structural resemblance to glutamate 1-semialdehyde.

Gabaculine resistance of Synechococcus glutamate 1-semialdehyde aminotransferase.

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.

Mechanism of glutamate semialdehyde aminotransferase. Roles of diamino- and dioxo-intermediates in the synthesis of aminolevulinate.

Glutamate semialdehyde aminotransferase was purified to homogeneity from pea leaves. The enzyme has an absorption spectrum with maxima at 345 and 416 nm. These chromophores were attributed to pyridoxamine phosphate and to pyridoxal phosphate bound as an aldimine respectively. Treatment of the enzyme with increasing concentrations of diaminovalerate produced a rapid fall in the 416 nm chromophore and a simultaneous increase in the 345 nm chromophore in a manner that indicated a stoichiometric reaction between 4,5-diaminovalerate and the pyridoxaldimine form of the enzyme. Treatment with 4,5-dioxovalerate produced the reverse reaction. The enzyme catalyzed the formation of aminolevulinate when dioxovalerate and diaminovalerate were present together and the maximal rate was 40% of that observed when glutamate semialdehyde itself was used as substrate. Conversion of the enzyme from its pyridoxaldimine to pyridoxamine form produced a proportional increase in activity towards glutamate semialdehyde, whereas reduction of the pyridoxaldimine form with sodium borohydride produced no change in this catalytic activity. It was concluded, therefore, that only the pyridoxamine form of the enzyme is active in catalyzing conversion of glutamate semialdehyde to aminolevulinate and that the catalytic mechanism includes enzyme-bound diaminovalerate as a central intermediate.

Characterization of glutamate‐1‐semialdehyde aminotransferase of Synechococcus

Synechococcus glutamate-1-semialdehyde aminotransferase was expressed in large amounts in transformed cells of Escherichia coli. The resulting purified enzyme has an absorption spectrum characteristic of B6-containing enzymes and could be converted to the pyridoxal-phosphate form with excess dioxovalerate (O2Val), and back to the pyridoxamine-phosphate form with diaminovalerate (A2Val). Both enzyme forms are similarly active in the conversion of glutamate 1-semialdehyde (GSA) to 5-aminolevulinate (ALev), suggesting that A2Val and O2Val are intermediates. Initial rates of ALev synthesis at various fixed concentrations of GSA followed typical Michaelis-Menten kinetics (Km of GSA for the pyridoxamine-phosphate form of GSA aminotransferase = 12 microM, kcat = 0.23 s-1). In submicromolar amounts A2Val stimulates ALev synthesis, and in a series of concentrations with various fixed concentrations of GSA, gives a family of parallel lines in Lineweaver-Burk plots (Km for A2Val = 1.0 microM). On the other hand, O2Val gives competitive inhibition of the pyridoxamine-phosphate form of GSA-aminotransferase and mixed-type inhibition of the pyridoxal-phosphate form (Ki for O2Val = 1.4 mM). In general the kinetics were typical of ping-pong bi-bi mechanisms in which A2Val is the second substrate (intermediate) and O2Val is an alternative first substrate. There is no compelling evidence that O2Val accepts an amino group at its C5 position resulting in the direct formation of ALev, or the reverse involving the apparent formation of O2Val from ALev. These results are consistent with the hypothesis that the mechanism of GSA aminotransferase mimics that of other aminotransferases and that A2Val is the intermediate.

Conditions for the assay of glutamate semialdehyde aminotransferase that overcome the problem of substrate instability

Investigations into the kinetic properties of glutamate semialdehyde aminotransferase, a key enzyme in the metabolic pathway leading to chlorophyll, are made difficult by the instability of the enzyme's substrate glutamate 1-semialdehyde. The rate of spontaneous disappearance of this compound from solution is shown to vary with the square of its concentration and to be pH-dependent. Thus using conditions appropriate to the assay of the enzyme, half of the substrate is lost from solution in a few minutes. Second-order rate constants for the reaction are determined and conditions are selected whereby the effects of the spontaneous reaction are rendered insignificant. The steady-state kinetic properties of the enzyme determined using these conditions are reported.

Separation and partial characterization of enzymes catalyzing δ-aminolevulinic acid formation in Synechocystis sp. PCC 6803

Formation of the universal tetrapyrrole precursor, δ-aminolevulinic acid (ALA), from glutamate via the five-carbon pathway requires three enzymes: glutamyl-tRNA synthetase, glutamyl-tRNA reductase, and glutamate-1-semialdehyde (GSA) aminotransferase. All three enzymes were separated from extracts of the unicellular cyanobacterium Synechocystis sp. PCC 6803, and two of them, glutamyl-tRNA synthetase and GSA aminotransferase, were partially characterized. After an initial high speed centrifugation and differential ammonium sulfate fractionation of cell extract, the enzymes were separated by successive affinity chromatography on Reactive Blue 2-Sepharose and 2′,5′-ADP-agarose. All three enzyme fractions were required to reconstitute ALA formation from glutamate. The apparent native molecular masses of glutamyl-tRNA synthetase and GSA aminotransferase were determined by gel filtration chromatography to be 63 and 98 kDa, respectively. Neither glutamyl-tRNA synthetase nor GSA aminotransferase activity was affected by hemin concentrations up to 10 and 30 μ m, respectively, and neither activity was affected by protochlorophyllide concentrations up to 2 μ m. GSA aminotransferase was inhibited 50% by 0.5 μ m gabaculine. The gabaculine inhibition was reversible for up to 1 h after its addition, if the gabaculine was removed by gel filtration before the enzyme was incubated with substrate. However, irreversible inactivation was obtained by preincubating the enzyme at 30 °C either for several hours with gabaculine alone or for a few minutes with both gabaculine and GSA. Neither pyridoxal phosphate nor pyridoxamine phosphate significantly affected the activity of GSA aminotransferase at physiologically relevant concentrations, and neither of these compounds reactivated the gabaculine-inactivated enzyme. It was noted that the presence of pyridoxamine phosphate in the ALA assay mixture produced a false positive color reaction even in the absence of enzyme.

Gabaculine-resistant glutamate 1-semialdehyde aminotransferase of Synechococcus. Deletion of a tripeptide close to the NH2 terminus and internal amino acid substitution

Glutamate 1-semialdehyde aminotransferase (GSA-AT) is the last enzyme in the C5 pathway converting glutamate into the tetrapyrrole precursor delta-aminolevulinate in plants, algae, and several bacteria. Sequence analysis of the genes encoding GSA-AT in barley, Synechococcus, and Escherichia coli revealed 50-70% similarity in the primary structures of the proteins. The enzyme is inhibited rapidly by gabaculine when added in approximately stoichiometric amounts with the enzyme. A gabaculine-tolerant Synechococcus strain, GR6, was found to produce a GSA-AT less sensitive to the inhibitor. Accordingly, the mutant gene was isolated and sequenced. In comparison with the wild-type gene it contains a deletion of nine nucleotides (position 12-20) and a guanine to adenine substitution (position 743). This resulted in the loss of the amino acids serine, proline, and phenylalanine (position 5-7) close to the NH2 terminus of the enzyme and an exchange of Met-248 for isoleucine in the middle of the polypeptide chain. Wild-type and mutant GSA-AT were expressed in E. coli and purified close to homogeneity. Although the specific activity of the mutant GSA-AT was only one-fifth of the wild type, it displayed a 100-fold increased resistance to gabaculine. Peaks in the absorption spectrum of the purified recombinant GSA-ATs at 335 and 417 nm are typical of a transaminase containing a B6 cofactor. Incubation with substrate and with inhibitor induced spectral changes characteristic of other gabaculine-sensitive, B6-requiring enzymes.

The Escherichia coli hemL gene encodes glutamate 1-semialdehyde aminotransferase

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.

Direct identification and quantification of the cofactor in glutamate semialdehyde aminotransferase from pea leaves

Glutamate asmialdehyde aminotransferase, a key enzyme in the synthetic pathway leading to chlorophyll was purified from pea, ( Pixum sativum) leaves. Although the preparation contained a single contaminant the enzyme could be unambiguously identified as a dimer of subunit molar mass 45 kDa having an absorption spectrum consistent with the presence of pyridoxamine phosphate as cofactor. The cofactor was released by treatment with strong phosphate at low pH and was identified and quantified fluorimetrically. The specific activity of the enzyme (1.4 μmol·min −1·mg −1; 23 nkatal·mg −1) is very much higher than previously reported.

Structural genes of glutamate 1-semialdehyde aminotransferase for porphyrin synthesis in a cyanobacterium and Escherichia coli

In bacteria 5-aminolevulinate, the universal precursor in the biosynthesis of the porphyrin nucleus of hemes, chlorophylls and bilins is synthesised by two different pathways: in non-sulphur purple bacteria (Rhodobacter) or Rhizobium 5-aminolevulinate synthase condenses glycine and succinyl-CoA into 5-aminolevulinate as is the case in mammalian cells and yeast. In cyanobacteria, green and purple sulphur bacteria, as in chloroplasts of higher plants and algae a three step pathway converts glutamate into 5-aminolevulinate. The last step is the conversion of glutamate 1-semialdehyde into 5-aminolevulinate. Using a cDNA clone encoding glutamate 1-semialdehyde aminotransferase from barley, genes for this enzyme were cloned from Synechococcus PCC6301 and Escherichia coli and sequenced. The popC gene of E. coli, previously considered to encode 5-aminolevulinate synthase, appears to be a structural gene for glutamate 1-semialdehyde aminotransferase. Domains with identical amino acid sequences comprise 48% of the primary structure of the barley, cyanobacterial and putative E. coli glutamate 1-semialdehyde aminotransferases. The cyanobacterial and barley enzymes share 72% identical residues. The peptide containing a likely pyridoxamine phosphate binding lysine is conserved in all three protein sequences.

Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.

Light Regulation of δ-Aminolevulinic Acid Biosynthetic Enzymes and tRNA in Euglena gracilis

Chlorophyll synthesis in Euglena, as in higher plants, occurs only in the light. The key chlorophyll precursor, delta-aminolevulinic acid (ALA), is formed in Euglena, as in plants, from glutamate in a reaction sequence catalyzed by three enzymes and requiring tRNA(Glu). ALA formation from glutamate occurs in extracts of light-grown Euglena cells, but activity is very low in dark-grown cell extracts. Cells grown in either red (650-700 nanometers) or blue (400-480 nanometers) light yielded in vitro activity, but neither red nor blue light alone induced activity as high as that induced by white light or red and blue light together, at equal total fluence rates. Levels of the individual enzymes and the required tRNA were measured in cell extracts of light- and dark-grown cells. tRNA capable of being charged with glutamate was approximately equally abundant in extracts of light- and dark-grown cells. tRNA capable of supporting ALA synthesis was approximately three times more abundant in extracts of light-grown cells than in dark-grown cell extracts. Total glutamyl-tRNA synthetase activity was nearly twice as high in extracts of light-grown cells as in dark-grown cell extracts. However, extracts of both light- and dark-grown cells were able to charge tRNA(Glu) isolated from light-grown cells to form glutamyl-tRNA that could function as substrate for ALA synthesis. Glutamyl-tRNA reductase, which catalyzes pyridine nucleotide-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA), was approximately fourfold greater in extracts of light-grown cells than in dark-grown cell extracts. GSA aminotransferase activity was detectable only in extracts of light-grown cells. These results indicate that both the tRNA and enzymes required for ALA synthesis from glutamate are regulated by light in Euglena. The results further suggest that ALA formation from glutamate in dark-grown Euglena cells may be limited by the absence of GSA aminotransferase activity.

Primary structure of a key enzyme in plant tetrapyrrole synthesis: glutamate 1-semialdehyde aminotransferase.

The formation of delta-aminolevulinate from glutamate 1-semialdehyde (GSA) is catalyzed by glutamate 1-semialdehyde aminotransferase (EC 5.4.3.8). The active form of the barley enzyme appears to be a dimer of identical subunits with a molecular mass of 46 kDa. From the purified enzyme, amino acid sequences of the N-terminal ends of the mature protein as well as an internal peptide were determined. DNA primers deduced from these peptide sequences were used to amplify with the polymerase chain reaction a cDNA sequence encoding part of the enzyme. Screening a cDNA library with this DNA fragment identified a full-length clone encoding the 49,540-Da precursor of the GSA aminotransferase. The transit peptide for chloroplast import consists of 34 amino acids. GSA aminotransferase and a precursor form were expressed on a multicopy plasmid in Escherichia coli. Both recombinant gene products reacted with an antibody against the barley GSA aminotransferase. Active barley GSA aminotransferase expressed in E. coli was shown to be active in assays of bacterial cell extracts. As a gene symbol for barley GSA aminotransferase, Gsa is proposed.

Cyanobacterial glutamate 1-semialdehyde aminotransferase: requirement for pyridoxamine phosphate

Glutamate 1-semialdehyde aminotransferase has been separated from metabolically related activities by gel filtration and affinity chromatography. The enzyme was inhibited by gabaculin, 4-amino 5-fluoropentanoic acid and pyridoxal 5-phosphate and stimulated by pyridoxamine 5-phosphate. The activity of enzyme recovered by elution after electrophoresis in non-denaturing polyacrylamide gels was wholly dependent on pyridoxamine 5-phosphate. A mechanism for the enzyme-catalysed reaction based on these observations is discussed.

Biosynthesis of Δ-aminolevulinate in Cyanidium caldarium: Characterization of tRNAGlu, ligase, dehydrogenase and glutamate 1-semialdehyde aminotransferase

The unicellular red algae Cyanidium caldarium converts the intact carbon skeleton of glutamate to δ-aminolevulinate. The components required for this conversion are: tRNAGlu, ligase, dehydrogenase, glutamate 1-semialdehyde aminotransferase, ATP, NADPH and Mg2+. The enzymes and the tRNA involved in δ-aminolevulinate synthesis were isolated from Cyanidium caldarium and characterised. Soluble proteins from greening cells were separated by gel filtration on Sephacryl S-300 and passed sequentially through Blue Sepharose, Matrex gel Red A and chlorophyllin Sepharose. The ligase and the dehydrogenase activities were bound to Blue Sepharose as well as Matrex gel red A. Chlorophyllin Sepharose retained the tRNAGlu, while glutamate 1-semialdehyde aminotransferase passed through the affinity columns and was collected in the run-off protein fraction. The run-off fraction also contained δ-aminolevulinate dehydratase and porphobilinogen deaminase. Glutamate was converted to glutamate 1-semialdehyde in the presence of tRNAGlu, ATP, NADPH and Mg2+, either by the Blue Sepharose-bound protein fraction or the Matrex gel Red A-bound protein fraction. In the presence of the aminotransferase-containing fraction, the glutamate 1-semialdehyde produced was converted into δ-aminolevulinate. Two compounds in addition to glutamate 1-semialdehyde and glutamyl tRNAGlu were produced from glutamate when the Blue Sepharose-bound protein fraction was incubated with tRNAGlu, ATP, NADPH and Mg2+. One of these was converted into δ-aminolevulinate upon incubation with the run-off protein fraction and was assumed to be an additional intermediate in the δ-aminolevulinate biosynthesis pathway. The tRNA preparation from Cyanidium caldarium gave a minor and a major peak of glutamate acceptor activity when analysed by High Pressure Liquid Chromatography on a C18 column containing trioctyl methylammonium chloride. The ability to convert glutamate to glutamate 1-semialdehyde was associated with the major peak of glutamate acceptor activity. Glutamate 1-semialdehyde aminotransferase of Cyanidium caldarium was stimulated by low concentrations (up to 100 μm) of pyridoxal 5′ phosphate and pyridoxamine 5′ phosphate. At higher concentrations (0.5 to 5mm), pyridoxamine 5′ phosphate was further stimulatory while pyridoxal 5′ phosphate was inhibitory. Gabaculine inhibited glutamate 1-semialdehyde aminotransferase and was more effective than the gabaculine-pyridoxal 5′ phosphate adduct, m-carboxyphenyl pyridoxamine 5′ phosphate.

A new method for the synthesis of glutamate 1-semialdehyde. Characterization of its structure in solution by NMR spectroscopy

Glutamate 1-semialdehyde is formed in high yields by ozonolysis of 4-vinyl-4-aminobutyric acid and purification of the reaction products by cation exchange chromatography. The structure of the product and its pyrrole derivative have been confirmed by1H-NMR and13C-NMR spectroscopy, and by its conversion to σ-aminolevulinate by purified glutamate 1-semialdehyde aminotransferase, and to uroporphyrinogen with porphobilinogen and uroporphyrinogen synthetase. The NMR spectra provide evidence for the presence in solution of a hydrate of glutamate 1-semialdehyde. Concentration dependent formation of dimers of the hydrate was also shown. It is suggested that chemical conversion of glutamate 1-semialdehyde to σ-aminolevulinate proceeds through dimeric intermediate stages.