CHARACTERIZATION OF AN ENDO-ACTING N-ACETYLGLUCOSAMINIDASE FROM CLOSTRIDIUM DIFFICILE Ryan Miller, Danielle Gutelius, Garrett Holmes, Zachary Suter, Joshua Jones, Kristen Hokeness, Christopher Reid, Department of Science and Technology, Bryant University, Smithfield, RI RI-INBRE Summer Undergraduate Research Fellowship Program Peptidoglycan (PG) is composed of alternating β-1,4N-acetylglucosamine (GlcNAc) and Nacetylmuramic acid (MurNAc), cross-linked by the peptide tetramer extending off of MurNAc, creating a meshed sheath-like structure that comprises the major structural component of the bacterial cell wall. This project characterizes the protein encoded by cd1034 from Clostridium difficile 630. The gene has been tentatively annotated as a mannosyl-glycoprotein endo-β-Nacetylglucosaminidase (GlcNAcase), and as an FlgJ homolog (Sphingomonas sp. A1). The protein CD1034 is predicted to cleave β-1,4 glycosidic linkages between GlcNAc and MurNAc, producing GlcNAc at the reducing end. Based on sequence alignment, CD1034 is classified in the Carbohydrate Acting Enzyme database (CAZy) as a member of glycosyl hydrolase family 73 (GH73). This family is poorly characterized, despite essential involvement in bacterial life. In characterizing this GlcNAcase, there is potential for elucidating the functions of similar enzymes in this family. Bacterial GlcNAcases function in a variety of roles such as septum degradation, toxin export, PG recycling, and flagellar assembly. By exploring GlcNAcases and elucidating mechanism of action, we can better understand this family of enzymes. Optimal expression conditions in Escherichia coli (BL21(DE3)pLysS) were found for CD1034d44, a CD1034 clone with an N-terminal His-tag lacking the N-terminal transmembrane domain. Purification from inclusion bodies was successful, yielding a near-homogenous sample of CD1034d44. In vitro reaction conditions were established by evaluating the effect of temperature, pH, and divalent cation requirements in turbidometric assays with purified PG.The role the PG stem peptide plays in substrate binding was explored using purified peptidoglycans of varying chemotype as well as synthetic substrates (GlcNAc-pNp).
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