Adaptations to the nitrate reductase from the deep-sea dwelling bacterium, Shewanella benthica

A project undertaken at the School of Chemistry, University of Sydney, and supervised by R. Codd

Organisms that reside permanently in environmental niches that are considered extreme (as defined from an anthropogenic viewpoint) are termed ‘extremophiles’ (‘extremus’ is ‘on the outside’). Metabolic flux in these organisms is maintained by subtle adaptations to their biomolecular machinery (proteins, polynucleotides, membranes) in order that molecular function is optimized under the particular environmental challenge: high or low temperatures (hyperthermophiles or psychrophiles, respectively), high pressure (barophiles or piezophiles), high salt (halophiles), high or low [H+] (acidophiles or alkaliphiles, respectively).
The principle focus of our research is upon the adaptation to metalloproteins and metal uptake and regulatory systems in extremophilic bacteria. The catalytic subunit of the enzyme target being studied in this project (periplasmic nitrate reductase) contains a molybdenum ion (or in some related enzymes, a tungsten ion) and an iron-sulfur cluster. Periplasmic nitrate reductase is a member of the dimethylsulfoxide (DMSO) reductase family of the mononuclear molybdenum-containing enzyme superfamily. These enzymes catalyse metabolically critical carbon-, nitrogen- and sulfur-based redox reactions in all organisms: archaea, bacteria, plants and humans. The substrates transformed by mononuclear molybdenum(tungsten) enzymes highlight the importance of these enzymes in the environment (substrates include: nitrate, dimethylsulfoxide, dimethylsulfide, trimethylamine N-oxide, selenate, arsenite and (per)chlorate) and in human biology (substrates include: sulfite and xanthine).

In this project, we aim to better understand the adaptations conferred upon periplasmic nitrate reductase, expressed by the deep-sea dwelling bacterium, Shewanella benthica (ATCC 43991), which was isolated in 1984 at a depth of 5,920 m from the Puerto Rico Trench, the deepest part of the Atlantic Ocean. The molecular mechanisms used by S. benthica to thrive under high pressure and in very cold waters have not been extensively studied.

Preliminary Results
Shewanella benthica is able to tolerate hydrostatic pressures up to 41400 kPa (about 400 times atmospheric pressure). As a facultative barophile, we have cultured the bacterium at atmospheric pressure under refrigerated (4 °C) conditions. Cultures do not grow at 20 °C. The cells, when transferred from 4 °C to room temperature burst (a pressure effect), which is useful to us in the context of protein purification. We are working towards optimizing the expression of native periplasmic nitrate reductase in S. benthica and the purification of the protein based on protocols developed in our laboratory for a related system (Figure 1). Ultimately, we aim to correlate the native enzyme properties to molecular level information of the protein obtained from homology modeling (sequencing of the S. benthica genome (strain KT99) is in progress (J. Craig Venter Institute)) or from crystallographic studies of the native protein.
Projected Outcomes
The mechanisms used by organisms to survive at thermal, pH, pressure and/or salt extremes are many and varied and provide an exciting and largely unexplored opportunity to transfer these mechanisms, perfected by Nature, into the realm of industrial biocatalysis. There is growing interest in the use of enzymes from cold-adapted organisms in technologies that would benefit from operation at modest conditions, such as cold-washing and in enantioselective reactions of relevance to pharmaceutics.
Pakchung, A. A. H.; Simpson, P. J. L.; Codd, R. (2006). Life on Earth. Extremophiles Continue to Move the Goal Posts. Environ. Chem. 3: 77-93.

Deming, J. W.; Hada, H.; Colwell, R. R.; Luehrsen, K. R.; Fox, G. E. (1984). The Ribonucleotide Sequence of 5S rRNA from Two Strains of Deep-Sea Barophilic Bacteria. J. Gen. Microbiol. 130: 1911-1920

DeLong, E. F.; Franks, D. G.; Yayanos, A. A. (1997). Evolutionary Relationships of Cultivated Psychrophilic and Barophilic Deep-Sea Bacteria. Appl. Envir. Microbiol. 63: 2105-2108.

Figure 1. Homology model of periplasmic nitrate reductase from a Shewanella species.