Characterization and Engineering of Fused Chorismate-Utilizing Enzymes

Howard University, Washington DC

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Abstract

DESCRIPTION (provided by applicant): In bacteria, the shikimate pathway-derived intermediate chorismate is an important branch point for the synthesis of a number of primary and secondary metabolites. Examples include the aromatic amino acids, folate, coenzymes Q and the antibiotics chloramphenicol and candicidin, to name a few. There are five known chorismate-utilizing enzymes that catalyze reactions in pathways that branch off from chorismate. Two of these are anthranilate synthase with its associated aminodeoxyisochorismate (ADIC) synthase activity, and p-aminobenzoic acid (PABA) synthase with its associated aminodeoxychorismate (ADC) activity. Most anthranilate syntheses involved in tryptophan biosynthesis have distinct TrpE and TrpG subunits;ADC synthase contains PabA and PabB subunits. The work proposed here is directed toward the characterization or engineering of fused chorismate-utilizing enzymes. One such enzyme is from Streptomyces venezuelae (Sv), a chloramphenicol-producing Gram positive bacterium. A second is from Streptomyces griseus, producer of the antifungal antibiotic candicidin (and streptomycin). A third originates in Escherichia coli. There are three major aims or goals: (1) the detailed characterization of the structure and function of the fused SvTrpEG, which, contrary to initial expectations, has been found to be an anthranilate synthase as opposed to an ADIC synthase;(2) the cloning, expression and preliminary characterization of PabAB from S. griseus;and (3) the engineering of a fused E. coli PabAB enzyme. Achievement of the first goal will provide a deeper understanding of what structurally and functionally sets TrpEG apart from the homologous phenazine biosynthetic enzyme PhzE. Achievement of goal 2 will further an understanding of how PabA and PabB subunits of the metabolically important enzyme PABA synthase associate and work together to produce ADC, from which PABA is formed through the catalytic action of a third enzyme, ADC lyase or PabC. It could also aid in the metabolic engineering of the candicidin biosynthetic pathway in order to increase yield of antibiotic. Finally, achievement of the third goal may solve a problem that has plagued the study of the interaction between the PabA and PabB subunits of E. coli PABA synthase: their weak interaction when separate.