Publications

• Glutamate 1-semialdehyde aminotransferase: anomalous enantiomeric reaction and enzyme mechanism.

  Smith M.A., Kannangara C.G., Grimm B.

  Glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes near 50% conversion of the racemic mixture of GSA to 5-aminolevulinate (ALA), indicating quantitative use of the L-glutamate-derived natural (S)-enantiomer as substrate. This enzymic reaction has been extensively studied with (R,S)-GSA b ... >> More

  Glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes near 50% conversion of the racemic mixture of GSA to 5-aminolevulinate (ALA), indicating quantitative use of the L-glutamate-derived natural (S)-enantiomer as substrate. This enzymic reaction has been extensively studied with (R,S)-GSA because it is readily purified in high yields following ozonolysis of racemic 4-vinyl-4-aminobutyric acid. However upon addition of (R,S)-GSA, GSA-aminotransferase is converted to the pyridoxal-P or internal aldimine form (418 nm) and not rapidly cycled back to the original pyridoxamine-P, as predicted by the rate of product (ALA) accumulation. Addition of the putative intermediate, (R,S)-4,5-diaminovalerate (DAVA), eliminates this rapid conversion of the enzyme by (R,S)-GSA to the internal aldimine and stimulates initial rates of ALA synthesis (2-3-fold) and results in corresponding increases in apparent equilibrium concentrations of ALA. These results indicate that DAVA is rate limiting and suggest anomalous reactivity of (R)-GSA. Steady-state and spectral kinetic experiments with individual purified enantiomers confirm anomalous reactivity of (R)-GSA: in the case of (S)-GSA, spectral changes are lesser in amplitude and at least 1 or 2 orders of magnitude more rapid. Only (S)-GSA yielded significant amounts of ALA. Since (R)-GSA is an apparent substrate in the first half-reaction, the resulting (R)-DAVA is either inactive or a poor substrate in the second half-reaction. << Less

  Biochemistry 31:11249-11254(1992) [PubMed] [EuropePMC]

• Glutamate-1-semialdehyde aminotransferase from Sulfolobus solfataricus.

  Palmieri G., Di Palo M., Scaloni A., Orru S., Marino G., Sannia G.

  Glutamate-1-semialdehyde aminotransferase (GSA-AT) from the extremely thermophilic bacterium Sulfolobus solfataricus has been purified to homogeneity and characterized. GSA-AT is the last enzyme in the C5 pathway for the conversion of glutamate into the tetrapyrrole precursor delta-aminolaevulinat ... >> More

  Glutamate-1-semialdehyde aminotransferase (GSA-AT) from the extremely thermophilic bacterium Sulfolobus solfataricus has been purified to homogeneity and characterized. GSA-AT is the last enzyme in the C5 pathway for the conversion of glutamate into the tetrapyrrole precursor delta-aminolaevulinate (ALA) in plants, algae and several bacteria. The active form of GSA-AT from S. solfataricus seems to be a homodimer with a molecular mass of 87 kDa. The absorption spectrum of the purified aminotransferase is indicative of the presence of pyridoxamine 5'-phosphate (PMP) cofactor, and the catalytic activity of the enzyme is further stimulated by addition of PMP. 3-Amino-2,3-dihydrobenzoic acid is an inhibitor of the aminotransferase activity. The N-terminal amino acid sequence of GSA-AT from S. solfataricus was found to share significant similarity with the eukaryotic and eubacterial enzymes. Evidence is provided that ALA synthesis in S. solfataricus follows the C5 pathway characteristic of plants, algae, cyanobacteria and many other bacteria. << Less

  Biochem. J. 320:541-545(1996) [PubMed] [EuropePMC]

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

  Pugh C.E., Harwood J.L., John R.A.

  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 enzy ... >> More

  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. << Less

  J Biol Chem 267:1584-1588(1992) [PubMed] [EuropePMC]

• Characterization of the different spectral forms of glutamate 1-semialdehyde aminotransferase by mass spectrometry.

  Brody S., Andersen J.S., Kannangara C.G., Meldgaard M., Roepstorff P., von Wettstein D.

  Glutamate 1-semialdehyde aminotransferase produces delta-aminolevulinate for the synthesis of chlorophyll, heme, and other tetrapyrrole pigments. The native enzyme from Synechococcus is pale yellow and has absorption maxima at 338 and 418 nm from vitamin B6. Yellow, colorless, and pink forms of th ... >> More

  Glutamate 1-semialdehyde aminotransferase produces delta-aminolevulinate for the synthesis of chlorophyll, heme, and other tetrapyrrole pigments. The native enzyme from Synechococcus is pale yellow and has absorption maxima at 338 and 418 nm from vitamin B6. Yellow, colorless, and pink forms of the protein are obtained by treatment with 4,5-dioxovalerate, 4,5-diaminovalerate, and acetylenic GABA, respectively. Compared to the native enzyme, the 418 nm absorption maximum in the yellow enzyme is enhanced and the 338 nm maximum reduced while the colorless enzyme has a heightened maximum at 338 nm and a barely detectable peak at 418 nm. The pink enzyme has an absorption maximum at 560 nm. When the native and colorless enzymes are repeatedly diluted in 0.5 M Na2HPO4, pH 7.0, and reconcentrated, pyridoxamine 5'-phosphate is released and the 338 nm maximum lost. Thus the 338 nm absorption maximum is associated with noncovalently bound pyridoxamine 5'-phosphate. NaBH4 reduction proved that the absorbance at 418 nm is from pyridoxal 5'-phosphate cofactor bound by a Schiff base to the protein. When the native, colorless, and yellow enzymes were subjected to electrospray ionization mass spectrometry, the B6 cofactor dissociated from the protein and gave a molecular weight of 46,401-46,418. Acetylenic GABA and NaBH4 were used for protein modification, and they reacted with the native and yellow enzymes but had no effect on the colorless enzyme. Pyridoxal 5'-phosphate bound covalently to the protein after NaBH4 reduction. Acetylenic GABA attached covalently to the enzyme produced an additional mass peak, 123-126 mass units higher, in the electrospray ionization spectrum.(ABSTRACT TRUNCATED AT 250 WORDS) << Less

  Biochemistry 34:15918-15924(1995) [PubMed] [EuropePMC]

• Crystal structure of glutamate-1-semialdehyde aminomutase: an alpha2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity.

  Hennig M., Grimm B., Contestabile R., John R.A., Jansonius J.N.

  The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an alpha2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-A resolution to an R-factor of 18.7% and good stereoche ... >> More

  The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an alpha2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-A resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme's unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel alpha-carboxylic acids, thereby suggesting a basis for the enzyme's reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153-181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153-181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153-181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153-181 remains. << Less

  Proc. Natl. Acad. Sci. U.S.A. 94:4866-4871(1997) [PubMed] [EuropePMC]