Collaborative Research: Evolution In Vitro: Structures of DAHPSynthase * KDOPSynthase Chimeras

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0110877 This project focuses on structural studies designed to reveal the mechanisms of action and of regulation of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) and 3-deoxy-D- manno-2-octulosonate-8-phosphate synthase (KDOPS) from E. coli. This work will complement mutational and biochemical analysis of the enzymes carried out collaboratively in the laboratory of departmental colleague and co-PI, Ronald Bauerle. DAHPS catalyzes the first step of aromaticbiosynthesis, the condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P). KDOPS catalyzes the condensation of PEP and D-arabinose-5-phosphate, an essential step in lipopolysaccharide biosynthesis. The two enzymes are distant homologs, possessing similar (beta/alpha)8 barrel folds. However, the three DAHPS isozymes differ from KDOPS in that they require a divalent metal for activation and are regulated by feedback inhibition, each being sensitive to a different one of the three aromatic amino acids. The following goals will be pursued during the project period: (1) to refine and analyze the crystal structure of DAHPS(Phe) complexed with Mn 2+, PEP and its feedback inhibitor, phenylalanine, and to compare the structure of this complex with those of structures already determined - [Mn*PEP], [Mn*PEP], [Mn*PGL], [Pb*PEP], and [Cd*PEP]; (2) to determine and refine the crystal structure of DAHPS(Trp)*Mn*PEP, which has already been crystallized; (3) to determine the crystal structures of DAHPS(Phe)*Mn complexed with analogs of PEP and of E4P, as indicated by the modeling studies of collaborator, M. Krauss; (4) to crystallize and determine the crystal structure(s) of mutant DAHPS(Phe) enzymes generated in Bauerle's lab whose characterictics are not consistent with our model of action and regulation; and (5) to crystallize and determine the crystal structure(s) of DAHPS/KDOPS chimeras generated in Bauerle's lab that have enzymatic activity.