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Two members of the phosphoenolpyruvate mutase (PEPM)/isocitrate lyase (ICL) superfamily were investigated to study their structure-function relationships and to identify sequence signatures that define a particular function. The first enzyme (PA4872) was a protein of unknown function from Pseudomonas aeruginosa. The second enzyme from Aspergillus niger (An07g08390) was thought to be an oxaloacetate acetyl hydrolase (OAH) because of its high sequence identity (∼60%) to an enzyme with confirmed OAH activity.
The X-ray crystal structure determination of PA4872 revealed unique features that guided the design of biochemical experiments, which ultimately led to the discovery that the enzyme is an oxaloacetate decarboxylase (OAD). Two structures of An07g08390, one with bound Mg2+ and the second with bound Mn2+ and the inhibitor 3,3-difluorooxaloacetate, were determined. The functional studies demonstrated that although the enzyme has OAH activity, it has a far better activity as a 2R,3S-dimethylmalate lyase (DMML). The active site structure of DMML indicated a proline residue (Pro240) as a marker of DMML function along with confirming the conserved locations of previously established signature residues for lyase activity.
OAD is the founding member of a family within the PEPM/ICL superfamily and thus defines the function of the remaining family members. However, the biological context in which OAD functions remains unknown. DMML is known to function in the nicotinate catabolism pathway but not all the members of the pathway are present in A. niger. Transcriptome analysis suggests that the DMML encoding gene is under carbon catabolite repression but the pathway in which the enzyme functions has not yet been identified.
Advisor: | Herzberg, Osnat |
Commitee: | Colombini, Marco, Culver, James, Herzberg, Osnat, Orban, John, Rokita, Steven |
School: | University of Maryland, College Park |
Department: | Molecular and Cell Biology |
School Location: | United States -- Maryland |
Source: | DAI-B 70/06, Dissertation Abstracts International |
Source Type: | DISSERTATION |
Subjects: | Biophysics |
Keywords: | Dimethylmalate lyase, Isocitrate lyase, Oxaloacetate decarboxylase, Phosphoenolpyruvate mutase |
Publication Number: | 3359411 |
ISBN: | 978-1-109-20214-4 |