Degree Type

Dissertation

Date of Award

2010

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Thomas A. Bobik

Abstract

Salmonella enterica is capable of utilizing 1,2-propanediol (1,2-PD) as a sole carbon and energy source in a coenzyme B12 (Adenosylcobalamin, AdoCbl) -dependent fashion that involves a bacterial microcompartment (MCP), the Pdu MCP. Pdu MCP is a polyhedral organelle composed entirely of protein subunits and its function is to sequester the intermediate propionaldehyde formed in the first step of 1,2-PD degradation in order to mitigate its toxicity and prevent DNA damage. Several sequentially-acting metabolic enzymes, including the diol dehydratase PduCDE and the propionaldehyde dehydrogenase PduP that respectively use AdoCbl and NAD+ as cofactors, are encapsulated within the solid protein shell of the MCP. During catalysis, the adenosyl-group of AdoCbl bound to PduCDE is periodically lost due to by-reactions and is usually replaced by a hydroxo-group resulting in the formation of an inactive OH-Cbl-enzyme complex, and the NAD+ is converted to NADH by PduP, which make it necessary to regenerate or/and replenish AdoCbl and NAD+ within the Pdu MCPs.

Recent crystallography studies suggested that some Pdu MCP shell proteins, such as PduA, T and U, have pores that may mediate the transport of enzyme substrates/cofactors across the MCP shell. However, it's possible that the cofactors might be regenerated in the local vicinity of the metabolic enzymes within the MCP, which could contribute to higher activity of the enzymes and thus to higher efficiency of 1,2-PD degradation.

In this work, two proteins encoded by the pdu locus consisting of genes specifically involved in 1,2-PD utilization, PduS and PduQ, were overexpressed, purified and characterized in vitro, and their in vivo fuctions were investigated as well.

Purified PduS enzyme exhibited the abilities to catalyze the two successive uni-electron reductions from cob(III)alamin to cob(I)alamin in vitro. The results indicated that PduS is a monomer and each monomer of PduS contains one non-covalently bound FMN and two [4Fe-4S] clusters which are oxygen-labile. Genetic studies showed that a pduS deletion decreased the growth rate of Salmonella on 1,2-PD supporting a role in cobalamin reduction in vivo. Further SDS-PAGE and Western blot of purified Pdu MCP and following MS-MS analysis demonstrated that the PduS protein is a component of the Pdu MCP. In addition, two-hybrid experiments indicated that PduS interacts with the PduO adenosyltransferase which is also located in the Pdu MCP and catalyzes the terminal step of AdoCbl synthesis.

Purified PduQ enzyme was identified as an oxygen-sensitive iron-dependent alcohol dehydrogenase (ADH) that catalyzes the interconversion between propionaldehyde and 1-propanol in vitro. The propionaldehyde reduction activity of PduQ was about 45 times higher than that of the 1-propanol oxidation at pH 7.0, indicating that this enzyme is more efficient for catalyzing aldehyde reduction in cells where approximatedly neutral pH is maintained. Kinetic studies indicated PduQ has a high affinity for the substrate and cofactor involved in this reduction reaction, which also suggest that the physiological function of PduQ is the reduction of aldehyde with the conversion of NADH to NAD+. The pduQ deletion impaired growth on 1,2-PD and led to a lower cell density, indicating that PduQ was required to support the maximal rate of 1,2-PD degradation by Salmonella. SDS-PAGE and Western blot of purified Pdu MCP and subsequent MS-MS analysis demonstrated that the PduQ protein is also associated with the Pdu MCP. Co-affinity purification illustrated a specific strong interaction between PduQ and PduP.

In conjunction with prior results, this work indicates that the Pdu MCP encapsulates a complete AdoCbl recycling system and that it is able to recycle the electron carrier NAD+ as well within the MCP lumen.

DOI

https://doi.org/10.31274/etd-180810-55

Copyright Owner

Shouqiang Cheng

Language

en

Date Available

2012-04-30

File Format

application/pdf

File Size

129 pages

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