Author(s): Trajan Nielson, Alexander Stewart
Mentor(s): Bruce R. Howard
Institution SUU
Our project aims to determine the three-dimensional structure of a halophilic malate synthase enzyme using X-ray crystallography. Malate synthase enzymes are essential members of the glyoxylate cycle and are classified as one of four isoforms based on protein chain length and sequence homologies. Isoform H is ~420 amino acid residues in length, isoform A is ~530 residues, isoform G is ~730 residues, and the fourth isoform is ~830 residues in length. Experimental structures have been determined for examples of isoform H, A, and G, but no experimentally determined structures have been reported for this fourth isoform. Our goal is to provide an experimentally determined structure for a member of this fourth isoform, specifically the malate synthase from Sulfolobus acidocaldarius. It has been reported to be maximally active at a temperature of 80 °C (176 °F). This enzyme has also been reported to not require a Mg2+ ion cofactor for activity, unlike all other isoforms of this enzyme. However, recent computer modeling of the enzyme structure using AlphaFold and RoseTTAFold suggests that all catalytic residues are conserved, arguing for a conserved mechanism requiring Mg2+. Our project aims to address this discrepancy and to verify the predicted structures by comparing them with an experimentally determined X-ray crystal structure. Having an experimentally confirmed structure will not only validate computational models but will also contribute to the database of known structures to improve future folding predictions. We will present progress made toward the overexpression of soluble S. acidocaldarius malate sythase in E. coli, the development of an effective purification strategy, and the eventual crystallization of this thermophilic enzyme.