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The equilibrium model (EM) (Daniel et al., 2001) postulates two forms of a folded enzyme, one catalytically active (Eact) and the other inactive (Einact), which interconvert via a fast thermal equilibrium (Keq) (Figure A). This model for enzyme catalysis accounts for experimentally observed time and temperature profiles of enzyme/substrate systems more accurately than the classically derived, single folded-species, model (Figure B). In both models, the denatured species (X) is formed via the kinact process, which is temperature and time-dependent. (FIgure A) (Figure B) Comparison between the equilibrium model and classical model for enzyme catalysis. For both, the vertical axis is catalytic rate (M s-1), the left-right axis is increasing temperature (K) and the back-front axis is assay duration (s). The physical basis for the Eact/Eina...
The VapBC toxin-antitoxin (TA) systems were first identified in 2005 and little is known about their contemporary biological function, despite the fact that VapBC TAs are the largest TA family and are widespread in bacteria and archaea (Arcus, Rainey, & Turner, 2005; Gerdes, Christensen, & Lobner-Olsen, 2005). Mycobacterium tuberculosis has a surprisingly large repertoire of 45 VapBC TAs. In contrast Mycobacterium smegmatis, a model organism for M. tuberculosis, contains only one vapBC operon, thereby making it an ideal system to uncover the possible role(s) that VapBC proteins play in mycobacteria. This thesis describes the functional characterisation of VapC from M. smegmatis and two homologues from Pyrobaculum aerophilum, along with biophysical characterisation of the VapBC complex from M. smegmatis. The VapBC proteins from M. smegm...
Interactions between proteins are a central concept in biology, and understanding and manipulation of these interactions is key to advancing biological science. Research into antibodies as customised binding molecules provided the foundation for development of the field of protein “scaffolds” for molecular recognition, where functional residues are mounted on to a stable protein platform. Consequently, the immunoglobulin domain has been describes as “nature’s paradigm” for a scaffold, and has been widely researched to make engineered antibodies better tools for specific applications. However, limitations in their use have lead to a number of non-immunoglobulin domains to be investigated as customisable scaffolds, to replace or complement antibodies. To be considered a scaffold, a protein domain must show an evolutionarily conserved hyd...
Toxin-antitoxin (TA) systems were identified more than 20 years ago on the mini F plasmid of Escherichia coli as plasmid stability elements; components responsible for purging bacterial cells that lack the plasmid from the population. More recent discovery of TA systems spanning a wide diversity of prokaryotic chromosomes, including that of Mycobacterium tuberculosis (M. tb), suggests a broader biochemical role. TA systems can be classified into a number of families, with the vapBC systems being by far the largest. The biochemical role of vapBC systems in M. tb remains unclear despite their abundance within the genome. This thesis describes the biochemical and functional characterisation of two vapBC systems encoded by operons Rv0065a/c and Rv0617a/c in the M. tb genome. VapCRv0617 overexpression had a bacteriostatic effect on the gro...
Proteins belonging to the PIN-domain family are widespread across bacteria, archaea and eukaryotes. PIN-domain structures are well characterised and contain three highly conserved acidic residues that are orientated to form the active site of the enzyme. VapC is the toxic component of a toxin-antitoxin (TA) complex, and contains a PIN-domain. The VapBC TAs are the largest TA family, and are found in expanded copy numbers in a variety of unrelated organism including the human pathogen Mycobacterium tuberculosis. It is proposed that TAs play a role in metabolic regulation, under conditions of stress. VapCs are metal dependent endoribonucleases that target specific sequences in single stranded RNA and inhibit translation by degrading mRNA transcripts. The mechanism by which VapC cleaves RNA and achieves this specificity is unknown. In...
Through ancestral sequence reconstruction (ASR) techniques, ancient enzymes can be recreated and biochemically tested, giving insight into the enzymes??? evolutionary history. A previous study by Hobbs et al. (2012) has shown that some ancestral 3-isopropylmalate dehydrogenase (IPMDH) enzymes of the Bacillus lineage are more catalytically efficient and kinetically stable than extant counterparts. Given these characteristics, this trend raises questions as to why ancestral Bacillus IPMDH enzymes have been superseded by catalytically slower and less kinetically stable counterparts. The homology between IPMDH and the dehydrogenases of tartrate, malate and isocitrate makes IPMDH an interesting model enzyme in terms of the evolution of substrate specificity. Here, the reconstruction of a 2.7 billion year old enzyme has been attempted ...
Extracellular nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that reduce the extracellular nucleotide signal and inactivate the purinogenic signalling pathway. These enzymes, in the presence of a divalent cation sequentially hydrolyses the ??- and ??- phosphoanhydride bond from a range of nucleotide di- or triphosphates to the corresponding nucleotide monophosphate. There is evidence that purinogenic signalling is present in higher plants and it is becoming clear that NTPDases play a role in the early stages of rhizobium infection during nodulation in legumes. To further our understanding of NTPDases, this study investigated the biochemical and structural characteristics of two legume NTPDases believed to be involved in nodulation. The first, 7WC, was isolated from the roots of white clover and the second, DbLnP,...
Thermophily has been proposed to be a primitive trait (Stetter, 2006, Pace, 1991, Woese, 1987) which has led to suggestions that all contemporary thermophilic species are the direct descendants of ancient thermophilic organisms. Ancestral sequence reconstruction (ASR) is a modern molecular technique which has been used to study the evolution of thermophily, however these studies have produced conflicting results. Studies utilising ASR to investigate the evolution of thermophily with two different proteins, elongation factor Tu and thioredoxin, have suggested a primitive origin for thermophily. However a recent study by Hobbs et al. (2012), in which ancestral IPMDH enzymes were reconstructed and biochemically characterised in order to investigate the evolution of thermophily in the Bacillus genus, suggested that thermophily may have evo...
Lsr2 is a small, basic DNA binding protein that is highly conserved in mycobacteria and related actinomycetes. Lsr2 is essential for growth in Mycobacterium tuberculosis and previous studies have shown that Lsr2 is involved in down-regulating a wide range of genes involved in cell wall synthesis and metabolic functions. This regulatory function is likely to influence bacterial growth and survival. This research investigated the biochemistry and 3D structure of Lsr2 from M. tuberculosis. Transmission electron microscopy (TEM) analysis of Lsr2 in complex with DNA revealed a regular fibril-like arrangement of protein coating double-stranded DNA. In addition, it was shown that Lsr2 physically protected DNA from DNase activity. The structure of the C-terminal DNA binding domain of Lsr2, determined by others part way through this research, ...
The rumen harbours a large and diverse microbial population that is responsible for the breakdown of plant material into smaller compounds, which can then be utilised by the animal. Butyrivibrio proteoclasticus is an anaerobic, Gram-positive bacterium originally isolated from the rumen of New Zealand cows. The entire genome of B. proteoclasticus has been sequenced. This revealed that a large proportion of the genome is devoted to polysaccharide degradation and reassembly1. Prior to the start of the research described in this thesis, 44 of the genes from the B. proteoclasticus genome annotated as being involved in fibre degradation had been cloned and expression of many has been tested. Two of these enzymes were expressed, purified and had had their 3D structures determined. Further characterisation of these two enzymes is presented her...
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