Lizelle Ann Piater

A Novel Chromate Reductase from Thermus scotoductus SA-01 Related to Old Yellow Enzyme

Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.

Reduction of U(VI) by the deep subsurface bacterium, Thermus scotoductus SA-01, and the involvement of the ABC transporter protein

In this study we investigated the effect of uranium on the growth of the bacterium Thermus scotoductus strain SA-01 as well as the whole cell U(VI) reduction capabilities of the organism. Also, site-directed mutagenesis confirmed the identity of a protein capable of a possible alternative mechanism of U(VI) reduction. SA-01 can grow aerobically in up to 1.25 mM uranium and has the capability to reduce low levels of U(VI) in under 20 h. TEM analysis performed on cells exposed to uranium showed extracellular and membrane-bound accumulation of uranium. The reductase-like protein was surprisingly identified as a peptide ABC transporter, peptide-binding protein. This study showcases the concept of protein promiscuity, where this protein with a distinct function in situ can also have the unintended function of a reactant for the reduction of U(VI).

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library

Aims:  The aim of this study was to demonstrate the application of environmental sample pre‐enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s).

A thioredoxin reductase-like protein from the thermophile, Thermus scotoductus SA-01, displaying iron reductase activity.

The transition metal iron is an important element for the sustenance of life--it can function either as an electron acceptor or as a donor and serves as a cofactor in many enzymes activities. The cytoplasmic NAD(P)H-dependent ferric reductase in Thermus scotoductus SA-01 shares high sequence and structural similarity to prokaryotic thioredoxin reductases. Here we report the sequence of the ferric reductase (which is typically annotated as a thioredoxin reductase-like protein) and a comparative kinetic study with the thioredoxin reductase from SA-01. Structurally, the most noteworthy difference, immediately apparent from the protein sequence, is the absence of the disulphide redox centre in the ferric reductase. This is the first report relating the attributes of such a redox protein to its ability to reduce a ferric substrate.

Metabolic promiscuity from the deep subsurface: a story of survival or superiority

The Witwatersrand Supergroup is a 2.9-billion-year-old formation of low permeability sandstone and shale with minor volcanic units and conglomerates with an ambient rock temperature of approximately 60°C. Thermus scotoductus SA-01 was isolated from fissure water at a depth of 3.2 kmbls in a South African gold mine and it shows the ability to reduce a variety of heavy metals under anaerobic conditions. It has been postulated that such microorganisms could play an important role in nutrient and metal cycling within the subsurface. Recently, our studies indicate that the cycling of metals could also occur under aerobic conditions and not only by the action of redox active enzymes, but other diverse metabolic proteins as well. In this study the capability of specific proteins to interact with metals is elucidated. Using Thermus SA-01 and its now completed genome sequence, metal reduction is studied through classic proteomic- and genomic methods. Finally we identify thermostable enzymes responsible for the transformation of various metals (Iron, Chrome, Uranium, Gold, etc) and discuss that reduction occurs via the serendipitous action of enzymes with other primary physiological functions, some of which are classical catabolic enzymes and anabolic proteins. This paper discusses the use of a ubiquitous enzyme/protein performing more than one function, possibly detoxifying the environment and using moonlighting as resource to decrease cellular energy requirements rather than elaborate metabolism in the subsurface.