Defense Date

6-21-2023

Graduation Date

Spring 5-10-2024

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Biological Sciences

School

School of Science and Engineering

Committee Chair

John F Stolz

Committee Member

Andrew VanDemark

Committee Member

Nancy Trun

Committee Member

Wook Kim

Keywords

outer membrane porin, RarA, Sulfurospirillum barnesii SES-3, arsenate, nitrite, AlphaFold, modeling, protein expression, E.coli Lemo21 DE3 cells

Abstract

Sulfurospirillum barnesii SES-3 is a gram-negative epsilon proteobacterium that grows anaerobically by coupling the oxidation of lactate to the reduction of arsenate. Previous work on metal and metalloid metabolism in S. barnesii had identified a redox active protein, designated RarA that when provided an artificial electron donor (i.e., reduced methyl viologen, FMN, or NADH), could reduce a variety of substrates, including arsenate, selenate, selenite, nitrite, and even phosphate. The enzyme’s inferred amino acid sequence showed weak similarity (30%) to outer membrane porins with homologs found in only certain species of Sulfurospirillum. Further, the enzyme did not contain cysteine residues or metal-binding motifs. In this study rarA was expressed in Escherichia coli strains heterologously and shown to have the same properties as the native protein in S. barnesii, conferring general metal/metalloid reductase activity. Heterologous expression of RarA in E.coli strains TG1 and BL21 with N terminus signal peptide was found to be toxic. However, deleting the signal peptide led to the protein being expressed in inclusion bodies when E.coli strains BL21(DE3) and Lemo21(DE3) expression systems were used. Soluble ‘RarA (‘denoting a N-terminus deletion) was achieved when expressed in E.coli Lemo21(DE3) at 16°C with low-speed agitation and 5 mM arsenate amended to the medium. Active ‘RarA was purified using a Ni-NTA column. The enzymatic kinetics of both the native protein and the expressed ‘RarA exhibited notable similarities, as evidenced by their Michaelis-Menten constants (Km) and maximum reaction rates (Vmax) for arsenate and nitrite. Specifically, the native protein showed a Km of 0.043 μM and a Vmax of 6.3μM min-1 for arsenate, and Km of 0.58 μM and a Vmax of 7.69 μM min-1 for nitrite. ‘RarA demonstrated a Km of 0.059 μM and a Vmax of 10 μM min-1 for arsenate and a Km of 0.061 μM and a Vmax of 10.3 μM min-1 for nitrite. The predicted mass of RarA was 55 kDa, and purification efforts to homogeneity lead to degradation into polypeptides of 30 kDa and 19 kDa, eventually losing their activity. Homology modeling of RarA provided compelling evidence that it is indeed an outer membrane porin belonging to the OprD family. The successful expression of active protein may facilitate elucidation of the mechanism of redox activity as well as the potential for bioremediation strategies (i.e,, arsenic, chromium).

Language

English

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