On the origin and evolution of thermophily: reconstruction of functional precambrian enzymes from ancestors of Bacillus.

Thermophily is thought to be a primitive trait, characteristic of early forms of life on Earth, that has been gradually lost over evolutionary time. The genus Bacillus provides an ideal model for studying the evolution of thermophily as it is an ancient taxon and its contemporary species inhabit a range of thermal environments. The thermostability of reconstructed ancestral proteins has been used as a proxy for ancient thermal adaptation. The reconstruction of ancestral "enzymes" has the added advantages of demonstrable activity, which acts as an internal control for accurate inference, and providing insights into the evolution of enzymatic catalysis. Here, we report the reconstruction of the structurally complex core metabolic enzyme LeuB (3-isopropylmalate dehydrogenase, E. C. 1.1.1.85) from the last common ancestor (LCA) of Bacillus using both maximum likelihood (ML) and Bayesian inference. ML LeuB from the LCA of Bacillus shares only 76% sequence identity with its closest contemporary homolog, yet it is fully functional, thermophilic, and exhibits high values for k(cat), k(cat)/K(M), and ΔG(‡) for unfolding. The Bayesian version of this enzyme is also thermophilic but exhibits anomalous catalytic kinetics. We have determined the 3D structure of the ML enzyme and found that it is more closely aligned with LeuB from deeply branching bacteria, such as Thermotoga maritima, than contemporary Bacillus species. To investigate the evolution of thermophily, three descendents of LeuB from the LCA of Bacillus were also reconstructed. They reveal a fluctuating trend in thermal evolution, with a temporal adaptation toward mesophily followed by a more recent return to thermophily. Structural analysis suggests that the determinants of thermophily in LeuB from the LCA of Bacillus and the most recent ancestor are distinct and that thermophily has arisen in this genus at least twice via independent evolutionary paths. Our results add significant fluctuations to the broad trend in thermal adaptation previously proposed and demonstrate that thermophily is not exclusively a primitive trait, as it can be readily gained as well as lost. Our findings also demonstrate that reconstruction of complex functional Precambrian enzymes is possible and can provide empirical access to the evolution of ancient phenotypes and metabolisms.

The molecular basis of the effect of temperature on enzyme activity.

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. The temperature midpoint (Teq) of the rapid equilibration between the two forms is related to the growth temperature of the organism, and the enthalpy of the equilibrium (DeltaHeq) to its ability to function over various temperature ranges. In the present study, we show that the difference between the active and inactive forms is at the enzyme active site. The results reveal an apparently universal mechanism, independent of enzyme reaction or structure, based at or near the active site, by which enzymes lose activity as temperature rises, as opposed to denaturation which is global. Results show that activity losses below Teq may lead to significant errors in the determination of DeltaG*cat made on the basis of the two-state ('Classical') model, and the measured kcat will then not be a true indication of an enzyme's catalytic power. Overall, the results provide a molecular rationale for observations that the active site tends to be more flexible than the enzyme as a whole, and that activity losses precede denaturation, and provide a general explanation in molecular terms for the effect of temperature on enzyme activity.

Determination of enzyme thermal parameters for rational enzyme engineering and environmental/evolutionary studies.

Of the two independent processes by which enzymes lose activity with increasing temperature, irreversible thermal inactivation and rapid reversible equilibration with an inactive form, the latter is only describable by the Equilibrium Model. Any investigation of the effect of temperature upon enzymes, a mandatory step in rational enzyme engineering and study of enzyme temperature adaptation, thus requires determining the enzymes' thermodynamic parameters as defined by the Equilibrium Model. The necessary data for this procedure can be collected by carrying out multiple isothermal enzyme assays at 3-5°C intervals over a suitable temperature range. If the collected data meet requirements for V max determination (i.e., if the enzyme kinetics are "ideal"), then the enzyme's Equilibrium Model parameters (ΔH eq, T eq, ΔG (‡) cat, and ΔG (‡) inact) can be determined using a freely available iterative model-fitting software package designed for this purpose.Although "ideal" enzyme reactions are required for determination of all four Equilibrium Model parameters, ΔH eq, T eq, and ΔG (‡) cat can be determined from initial (zero-time) rates for most nonideal enzyme reactions, with substrate saturation being the only requirement.