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Title: Structural studies on dehydrogenases
Author: Elbrghathi, Abdelhamid
ISNI:       0000 0004 5356 4803
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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The amino acid dehydrogenase superfamily has good potential for biotechnological use for example, either as enzymes for the production of non-natural amino acids, or for use as biosensors. Despite many years work on this family, there is still a gap understanding aspects of both substrate and cofactor binding and reaction mechanism. The crystal structures of M. smegmatis Glutamate dehydrogenase (GluDH) have been determined in two crystal forms, one of a ternary complex (GluDH/NADP+/2oxoglutarate) at 1.64A, and one of a binary complex (GluDH/NADPH) at 1.78A. The binary complex structures of C. symbiosum GluDH, with NAD+ and L-glutamate have also been solved at 1.80 A and 1.24A respectively. Comparison of these structures has allowed the nature of coenzyme specificity in GluDHs to be structurally characterized. Analysis of M. smegmatis GluDH/NADP+/2oxoglutarate ternary complex showed that 2-oxoglutarate bound to the enzyme as the gem-diol in an extended conformation, which mimics the structure of the carbinolamine intermediate. This structure, together with the high resolution C. symbiosum GluDH/L-glutamate complex structure, where the substrate binds in a compact, curved conformation have given valuable insights into the progress of the reaction. They show that the substrate alters its conformation during the reaction, explaining how the abortive reduction of the keto acid to the hydroxy acid is prevented, and showing the stereochemistry of attack by ammonia on the keto acid substrate, aspects of the mechanism that have been poorly understood. In addition, the structure of the AADH family member Bacillus sphaericus phenylalanine dehydrogenase in complex with NADH has been determined. Furthermore, initial experiments towards the structure of mycobacterium smegmatis typeII NADH: menaquinone oxidoreductase, which is a potential anti bacterial drug target are described.
Supervisor: Baker, Patrick Julian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available