Defense Date

6-30-2011

Graduation Date

Summer 2011

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Partha Basu

Committee Member

Michael Cascio

Committee Member

Mihaela Rita Mihailescu

Committee Member

John F. Stolz

Keywords

Campylobacter, Heterologous expression, Molybdenum enzymes, Nitrate reductase, Periplasmic, Protein purification

Abstract

In this study the nitrate metabolism of Campylobacter jejuni and Sulfurospirillum barnesii will be examined, specifically the periplasmic nitrate reductase (Nap) enzyme which is transforms of nitrate to nitrite. The catalytic subunit, NapA, is a molybdenum dependent enzyme. Isolation of molybdenum containing enzymes is not straightforward as co-factor can be lost during protein purification procedures. This study used two protein purification methods to isolate NapA. First, NapA was isolated directly from S. barnesii using protein fractionation and anion exchange chromatography. Second, molecular cloning was used to express the recombinant affinity-tagged S.barnesii and C. jejuni NapA proteins from E. coli. Immobilized metal affinity chromatography was used to isolate the recombinant proteins. NapD was co-expressed with NapA to aid in post-translational modifications. The reduced methyl viologen assay was used to study the kinetics of nitrate reduction. Comparison of the native and recombinant NapA kinetic properties suggests that the recombinant enzyme have attenuated activity. The theoretical structure of C. jejuni NapA was calculated using homology modeling techniques. Comparison of the C. jejuni NapA with structures of NapA from other organisms indicates that C. jejuni NapA has large sequence inserts on the outside of the protein. Furthermore, the napA operon of C. jejuni and S. barnesii display distinct gene content and organization.

Format

PDF

Language

English

Download

Share

COinS