published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.

The formation of bacterial biofilms requires an extracellular matrix to facilitate adherence of bacteria to the surface they colonize. Carbohydrate polymers, known as exopolysaccharides, form a key component of most biofilm matrices. A wide variety of medically-important biofilm forming bacterial strains, including S. epidermidis, S. aureus, E. coli, B. pertussis, and Y. pestis generate the same β-1,6-N-acetyl glucosamine (PNAG) homopolymer as a key biofilm matrix exopolysaccharide. In E. coli, as well as in the other bacterial strains, the PNAG undergoes partial enzymatic de-N-acetylation, which is essential for surface attachment and subsequent biofilm formation. In vivo studies implied that the enzyme responsible for carrying out de-N-acetylation in E. coli is PgaB, an enzyme with sequence homologues in many Gram negative species capable of forming biofilms. In this work, the first biochemical characterization of PgaB is presented. We confirmed the activity of PgaB on β-1,6-GlcNAc oligosaccharides. The activity of PgaB is specific for the β-1,6 linkage and no de-N-acetylation of β-1,4-GlcNAc oligosaccharides was detected. Enzyme activity is dependent on the degree of substrate polymerization, as the second order rate constant for pentasaccharide substrate was determined to be four times higher than that of the tetrasaccharide substrate. Oligosaccharide sequencing studies indicate that there may be a pattern in the de-N-acetylation of substrates by PgaB. The central residue is modified in mono-de-N-acetylated pentasaccharide substrate, while di-de-N-acetylated hexasaccharide substrate shows modification mainly at the third and fifth residues from the non-reducing terminus of the substrate. Activity studies revealed that PgaB is activated by Ni2+ as well as by Fe2+, which is uncommon for deacetylase enzymes. Metal coordination to active site residues His184 and His189 was confirmed by mutagenesis studies, which also indicated that the metal likely plays a catalytic role. The results of these metal dependence studies support the observed binding of nickel and iron to the active site in PgaB crystal structures. The characterization studies presented in this thesis allow us to gain a better understanding of the de-N-acetylation aspect of the PNAG biosynthetic process and will serve as a basis for enzyme inhibitor design.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Ulm, Univ., Diss., 2007.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 105-114) Also available in print.

Thesis (Ph. D. in Biochemistry)--Vanderbilt University, Aug. 2008. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 104-115).

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 140 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 119-140).