A new intrinsic thermal parameter for enzymes reveals true temperature optima

Two established thermal properties of enzymes are the Arrhenius activation energy and thermal stability. Arising from anomalies found in the variation of enzyme activity with temperature, a comparison has been made of experimental data for the activity and stability properties of five different enzymes with theoretical models. The results provide evidence for a new and fundamental third thermal parameter of enzymes, Teq, arising from a subsecond timescale-reversible temperature-dependent equilibrium between the active enzyme and an inactive (or less active) form. Thus, at temperatures above its optimum, the decrease in enzyme activity arising from the temperature-dependent shift in this equilibrium is up to two orders of magnitude greater than what occurs through thermal denaturation. This parameter has important implications for our understanding of the connection between catalytic activity and thermostability and of the effect of temperature on enzyme reactions within the cell. Unlike the Arrhenius activation energy, which is unaffected by the source (“evolved”) temperature of the enzyme, and enzyme stability, which is not necessarily related to activity, Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological, and environmental aspects of the adaptation of life to temperature in a way that has not been described previously. We may therefore expect the effect of evolution on Teq with respect to enzyme temperature/activity effects to be more important than on thermal stability. Teq is also an important parameter to consider when engineering enzymes to modify their thermal properties by both rational design and by directed enzyme evolution

The equilibrium model for the effect of temperature on enzymes: Insights and implications

A new, experimentally-validated “Equilibrium Model” describes the effect of temperature on enzymes, and provides a new mechanism for the reversible loss of enzyme activity with temperature. It incorporates two new, fundamental parameters that allow a complete description of the effect of temperature on enzyme activity: ΔHeq and Teq. ΔHeq emerges as an intrinsic and quantitative measure of enzyme eurythermal adaptation, while Teq, the equilibrium temperature, has fundamental and technological significance for our understanding of the effect of temperature on enzymatic reactions. For biotechnological purposes, these parameters need to be considered when enzymes are applied or engineered for activity at high temperatures

Promiscuity in the part-phosphorylative Entner–Doudoroff pathway of the archaeon Sulfolobus solfataricus

AbstractThe hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a ‘promiscuous’ non-phosphorylative variant of the Entner–Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner–Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of d-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose

Structurally-informed Mutagenesis of a Stereochemically Promiscuous Aldolase Produces Mutants that Catalyse the Diastereoselective Syntheses of all Four Stereoisomers of 3-Deoxy-Hexulosonic acid

[Image: see text] A 2-keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus catalyzes the nonstereoselective aldol reaction of pyruvate and d-glyceraldehyde to produce 2-keto-3-deoxygluconate (d-KDGlc) and 2-keto-3-deoxy-d-galactonate (d-KDGal). Previous investigations into curing the stereochemical promiscuity of this hyperstable aldolase used high-resolution structures of the aldolase bound to d-KDGlc or d-KDGal to identify critical amino acids involved in substrate binding for mutation. This structure-guided approach enabled mutant variants to be created that could stereoselectively catalyze the aldol reaction of pyruvate and natural d-glyceraldehyde to selectively afford d-KDGlc or d-KDGal. Here we describe the creation of two further mutants of this Sulfolobus aldolase that can be used to catalyze aldol reactions between pyruvate and non-natural l-glyceraldehyde to enable the diastereoselective synthesis of l-KDGlc and l-KDGal. High-resolution crystal structures of all four variant aldolases have been determined (both unliganded and liganded), including Variant 1 with d-KDGlc, Variant 2 with pyruvate, Variant 3 with l-KDGlc, and Variant 4 with l-KDGal. These structures have enabled us to rationalize the observed changes in diastereoselectivities in these variant-catalyzed aldol reactions at a molecular level. Interestingly, the active site of Variant 4 was found to be sufficiently flexible to enable catalytically important amino acids to be replaced while still retaining sufficient enzymic activity to enable production of l-KDGal

Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in <em>Geobacillus thermoglucosidasius </em>

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcoholviaan aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD+pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophileGeobacillus thermoglucosidasiushas been determined to 2.5 Å resolution. This is the first structure to be reported for such a domain.In silicomodelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.</jats:p

A novel β-xylosidase structure from Geobacillus thermoglucosidasius:The first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

Geobacillus thermoglucosidasiusis a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding aG. thermoglucosidasiusβ-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed inEscherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme–substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of theG. thermoglucosidasiusβ-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.</jats:p

Econometric forecasts for Scotland

In the first issue (July 1975) of the Fraser of Allander Institute Quarterly Economic Commentary, the special article, by Professor J McGilvray, reviewed the problems associated with constructing regional econometric models to forecast key economic variables. Since that time, a number of forecasts for the Scottish economy have been made in the main text or in special articles of the Quarterly Commentary. Many of these have been underpinned by forecasting relationships which have been estimated for particular sectors of the economy. Up to now we have been unable to produce a set of relationships which could genuinely be described as a 'model' of the Scottish economy. The reason for this is simple, but illustrative of the type of problem discussed by Professor McGilvray. To understand it one must be acquainted with the fundamental differences which exist between national and regional economic models

Crystal structure of an inferred ancestral bacterial pyruvate decarboxylase

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a key enzyme in homofermentative metabolism where ethanol is the major product. PDCs are thiamine pyrophos­phate- and Mg2+ ion-dependent enzymes that catalyse the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. As this enzyme class is rare in bacteria, current knowledge of bacterial PDCs is extremely limited. One approach to further the understanding of bacterial PDCs is to exploit the diversity provided by evolution. Ancestral sequence reconstruction (ASR) is a method of computational molecular evolution to infer extinct ancestral protein sequences, which can then be synthesized and experimentally characterized. Through ASR a novel PDC was generated, designated ANC27, that shares only 78% amino-acid sequence identity with its closest extant homologue (Komagataeibacter medellinensis PDC, GenBank accession No. WP_014105323.1), yet is fully functional. Crystals of this PDC diffracted to 3.5 Å resolution. The data were merged in space group P3221, with unit-cell parameters a = b = 108.33, c = 322.65 Å, and contained two dimers (two tetramer halves) in the asymmetric unit. The structure was solved by molecular replacement using PDB entry 2wvg as a model, and the final R values were Rwork = 0.246 (0.3671 in the highest resolution bin) and Rfree = 0.319 (0.4482 in the highest resolution bin). Comparison with extant bacterial PDCs supports the previously observed correlation between decreased tetramer interface area (and number of interactions) and decreased thermostability

Draft genome sequences of Geobacillus sp. Strains CAMR5420 and CAMR12739

Thermophilic Geobacillus spp. can efficiently hydrolyse hemicellulose polymers and are therefore of interest in biotechnological applications. Here we report the genomes of two hemicellulotyic strains, Geobacillus sp. CAMR12739 and CAMR5420.Genomics Research Institute, University of Pretoriahttp://genomea.asm.orghb201