On the Origin and Evolution of Thermophily: Reconstruction of Functional Precambrian Enzymes from Ancestors of Bacillus

Abstract

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.

Reconstructed Ancestral Enzymes Impose a Fitness Cost upon Modern Bacteria Despite Exhibiting Favourable Biochemical Properties

“…The ancestral inference and reconstruction of ANC1, ANC2, ANC3 and ANC4 (Hobbs et al 2012) and LeuB S-aw and LeuB S-unaw (Groussin et al 2015) have been reported previously. In brief, the sequences of ANC1, ANC2, ANC3 and ANC4 were inferred under the maximum likelihood criterion using a phylogeny of Bacillus species based on LeuB protein sequences and the LG substitution model implemented in PAML (Yang 2007).…”

“…For example, Hart et al (2014) recently reconstructed ancestors of ribonuclease H1 (RNH) from two lineages of bacteria, one mesophilic and one thermophilic, and found that the ancestors exhibited intermediate thermostabilities between those of contemporary RNHs from Escherichia coli and Thermus thermophilus. Furthermore, in our previous study of LeuBs from the Bacillus genus (Hobbs et al 2012), one of our inferred ancestral LeuBs (ANC2) exhibited a psychrophilic/mesophilic T opt , low DG z NÀU and only moderate k cat .…”

“…Since the first ancestral reconstructions by Malcolm et al (1990) and Stackhouse et al (1990), there have now been more than 40 published ASR studies and two common properties exhibited by inferred ancestral proteins/enzymes have emerged: (i) high stability-both thermostability (usually measured as the temperature midpoint of thermal denaturation, T m , or optimum temperature for activity, T opt ) and kinetic stability (measured as the free energy for unfolding, DG z NÀU )- (Akanuma et al 2013;Butzin et al 2013;Gaucher et al 2003Gaucher et al , 2008Groussin et al 2015;Hobbs et al 2012;Miyazaki et al 2001;Perez-Jimenez et al 2011;Risso et al 2013;Watanabe et al 2006a;Watanabe and Yamagishi 2006b) k cat and (ii) high catalytic activity and/or catalytic efficiency (usually expressed as k cat and k cat / K M , respectively) (Stackhouse et al 1990;Akanuma et al 2013;Butzin et al 2013;Groussin et al 2015;Hobbs et al 2012;Perez-Jimenez et al 2011;Risso et al 2013;Watanabe and Yamagishi 2006b;Jermann et al 1995). These increases in stability and catalytic activity compared with contemporary proteins/enzymes can be dramatic, for example Risso et al (2013) reported that their inferred ancestral b-lactamases were more stable than their contemporary descendants by as much as 35°C, and Perez-Jimenez et al (2011) found that their reconstructed ancestral thioredoxins displayed catalytic rate constants up to 30-fold higher than modern thioredoxins at pH 5, as well as being up to 32°C more stable.…”

“…These increases in stability and catalytic activity compared with contemporary proteins/enzymes can be dramatic, for example Risso et al (2013) reported that their inferred ancestral b-lactamases were more stable than their contemporary descendants by as much as 35°C, and Perez-Jimenez et al (2011) found that their reconstructed ancestral thioredoxins displayed catalytic rate constants up to 30-fold higher than modern thioredoxins at pH 5, as well as being up to 32°C more stable. We have previously used ASR to reconstruct presumed ancestral sequences of the core metabolic enzyme LeuB (3-isopropylmalate dehydrogenase, IPMDH, EC 1.1.1.85) from the Bacillus genus (Hobbs et al 2012) and the Firmicutes phylum (Groussin et al 2015), and also observed enhancements in stability and activity. In our Bacillus study, three out of the four reconstructed LeuBs were thermophilic (represented by high T opt and T m values) and exhibited k cat values C2-fold higher than that exhibited by the contemporary B. subtilis LeuB enzyme (Hobbs et al 2012).…”

“…We have previously used ASR to reconstruct presumed ancestral sequences of the core metabolic enzyme LeuB (3-isopropylmalate dehydrogenase, IPMDH, EC 1.1.1.85) from the Bacillus genus (Hobbs et al 2012) and the Firmicutes phylum (Groussin et al 2015), and also observed enhancements in stability and activity. In our Bacillus study, three out of the four reconstructed LeuBs were thermophilic (represented by high T opt and T m values) and exhibited k cat values C2-fold higher than that exhibited by the contemporary B. subtilis LeuB enzyme (Hobbs et al 2012). The reconstructed enzyme ANC4 (which sits at the base of the Bacillus tree; Fig.…”