Studies on Glutamate 1-Semialdehyde Aminotransferase

Callaghan, Josephine Felicity Anna (1998) Studies on Glutamate 1-Semialdehyde Aminotransferase. University of Southampton, Doctoral Thesis.

Abstract

Glutamate1-semialdehyde aminotransferase (GAS.AT), catalyses the conversion of glutamate 1-semialdehyde (GSA) into 5-aminolaevulinic acid (ALA) and represents the final stage of the C-5 pathway for ALA synthesis that is found in a variety of photo- and non-photosynthetic bacteria as well as in algae and higher plants.

In common with other aminotransferases, GSA.AT requires the coenzyme pyridoxal 5'-phosphate and catalyses a ping-pong mechanism which can follow one of two routes, depending on whether the enzyme is initially in the pyridoxamine 5'-phosphate (PMP) or in the pyridoxal 5-phosphate (PLP) form. The PMP form of the enzyme reacts with GSA to yield 4,5-diaminovaleric acid (DAVA) as an intermediate in the first part of the reaction whereas in the PLP form dioxovaleric acid (DOVA) is generated. The intermediates are used in the second part of the reaction to produce ALA and to regenerate the original form of the enzyme.

GSA.AT has been highly purified from recombinant strains of Escherichia coli and Synechococcus harbouring the gene encoding the enzyme and from native Arabidopsis thaliana. The resulting GSA.ATs have been characterized and their properties and mechanistic routes investigated under absorption spectrometry, laser desorbtion mass spectrometry, electrospray mass spectrometry, thin layer chromatography and steady state kinetics. Spectroscopic studies follow the changes of the cofactor from PLP to PMP forms, or vice versa, during aminotransferase half reactions.

Substrate analogues, including the lower homologue, aspartate 1-semialdehyde (ASA) was able to act as a partial substrate to form diaminobutyric acid (DABA). Potential mechanism-based inhibitors of the enzyme, including reduced forms of GSA and ALA were also studied. These substrate analogues gave interesting spectroscopic findings and formed a basis for the design of inhibitors for use as potential bactericides and herbicides.

Preliminary attempts in the crystallisation of GSA.AT from E. coli have resulted in the production of crystals and comparative studies with structure of the GSA.AT from Synechococcus GSA.AT are under way.

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