Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases

Abstract

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.

Keywords: ADP-dependent kinase; ancestral enzymes; archaea; crystal structure; glucokinase; phosphofructokinase; protein evolution; substrate specificity.

Figure 1.

Dendrogram of the ADP-dependent kinases family and Euryarcheotas organisms. A, consensus phylogenetic tree of the ADP-dependent sugar kinases family obtained by Bayesian inference using protein sequences. Nodes of resurrected ancestral enzymes are indicated as colored circles: ancGK/PFK in black, ancMMT in green, ancPFK-MT in pink, ancPFK-M in purple, and ancPFK-T in red. The following current enzymes are shown in their respective groups: glucose-specific from T. litoralis (TlGK), the specific by fructose-6-P from P. horikoshii (PhPFK), and the bifunctional from M. jannaschii (MjPFK/GK). B, species tree adapted from TimeTree (19) and Petitjean et al. (18), dotted lines show possible horizontal gene transfer events.

Figure 2.

Trace of the kinetic parameters of PFKs specificity en route to Thermococcales (A) and Methanococcales (B) using glucose or fructose-6-P as substrates. Divergence times were estimated by the RelTime method implemented in Mega7, using the speciation events shown in Fig. 1B (ancGK/PFK 3.59 Ga and ancMMT 3.47 Ga) as calibration points. The estimated times were: ancMT 1.98, ancM 1.51, and ancT 0.93 Ga. Evolution of K_m and k_cat shows that the main change toward fructose-6-P specificity in Thermococcales occurred in the transition between ancestors ancMT (1.98 Ga) and ancT (0.93 Ga), and was mainly caused by a loss in glucose affinity together with a concomitant increase in fructose-6-P affinity, leading to a 6 × 10⁴-fold increment in fructose-6-P specificity.

Figure 3.

Crystallographic structure of ancMT. A, open and semi-closed conformation of the ancMT structure, the open conformation corresponds to chain A (in red) and the semi-closed conformation to chain B (in blue). The r.m.s. deviation values for the whole protein and for the small domain are 1.2 and 2.5 Å, respectively. B, ancMT colored according to secondary structure: α-helices in red, β-sheet strands in yellow, and loops in green. Surface of the small domain is colored in gray and that of the large domain in white. C, AMP-binding site, the phosphate group of AMP appears in two positions due to conformational heterogeneity. Water molecules are labeled as w and the mF_o − DF_c omit map is contoured at 3σ for AMP. Yellow dotted lines indicate h-bonds calculated with PyMOL.

Figure 4.

Molecular modeling of fructose-6-P and glucose in the active site of the ancMT, ancM, and ancT ancestors. Oxygen atoms are colored in red, carbons in green, nitrogens in blue, and polar hydrogens are showed in white. The only change found at the active site of these ancestors (E to A) is highlighted in bold. Yellow dotted lines indicate h-bonds calculated with PyMOL.

Figure 5.

Nonbonding energy between ancMT and β-d-fructose-6-P or β-d-glucose calculated from molecular dynamic simulations. Simulations of 30 ns were performed for the Mg-ADP complex. The average nonbonding energy (Van der Waals plus electrostatic interactions) for all residues with at least one atom within 5 Å from the substrate was calculated. Only residues with over 1 kcal/mol are shown. The averaged interactions with positive energy values are shown in red and negative values are shown in black. Both simulations were performed with the force-field CHARMM 27 and NAMD 2.8 software. Error bars represent S.D. from 1500 frames. Residue conservation in sequence alignment of current enzymes (bifunctional and fructose-6-P-specific enzymes from Methanococcales and Thermococcales, respectively) is shown as sequence logo.

Figure 6.

Glucose saturation curves for ancMT and its E72A mutant. Saturation curve for ancMT was fitted to the substrate inhibition equation. For the E72A mutant saturation was not observed even at glucose concentrations higher than 600 mm. Inset, activity of the E72A mutant with glucose as substrate in a different velocity scale. The activity shows a linear dependence on glucose concentration and the k_cat/K_m value was obtained from the slope, considering that glucose concentrations are below the K_m value. Error bars represent S.E. from three independent measurements.

Figure 7.

Maximum likelihood reconstruction of substrate utilization as discrete binary characters. The left tree of the figure shows the results for the use of glucose, and on the right those corresponding to the use of fructose-6-P. The circles on leaf nodes and on ancestors represent a pie chart where black is the probability of use of the particular substrate (glucose or fructose-6-P) and white the probability of it not being used. Gray circles indicate out-group sequences used in the analysis.