Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul;25(7):1319-31.
doi: 10.1002/pro.2904. Epub 2016 Mar 24.

Gradual Neofunctionalization in the Convergent Evolution of Trichomonad Lactate and Malate Dehydrogenases

Affiliations
Free PMC article

Gradual Neofunctionalization in the Convergent Evolution of Trichomonad Lactate and Malate Dehydrogenases

Phillip A Steindel et al. Protein Sci. .
Free PMC article

Abstract

Lactate and malate dehydrogenases (LDH and MDH) are homologous, core metabolic enzymes common to nearly all living organisms. LDHs have evolved convergently from MDHs at least four times, achieving altered substrate specificity by a different mechanism each time. For instance, the LDH of anaerobic trichomonad parasites recently evolved independently from an ancestral trichomonad MDH by gene duplication. LDH plays a central role in trichomonad metabolism by catalyzing the reduction of pyruvate to lactate, thereby regenerating the NAD+ required for glycolysis. Using ancestral reconstruction methods, we identified the biochemical and evolutionary mechanisms responsible for this convergent event. The last common ancestor of these enzymes was a highly specific MDH, similar to modern trichomonad MDHs. In contrast, the LDH lineage evolved promiscuous activity by relaxing specificity in a gradual process of neofunctionalization involving one highly detrimental substitution at the "specificity residue" (R91L) and many additional mutations of small effect. L91 has different functional consequences in LDHs and in MDHs, indicating a prominent role for epistasis. Crystal structures of modern-day and ancestral enzymes show that the evolution of substrate specificity paralleled structural changes in dimerization and α-helix orientation. The relatively small "specificity residue" of the trichomonad LDHs can accommodate a range of substrate sizes and may permit solvent to access the active site, both of which promote substrate promiscuity. The trichomonad LDHs present a multi-faceted counterpoint to the independent evolution of LDHs in other organisms and illustrate the diverse mechanisms by which protein function, structure, and stability coevolve.

Keywords: ancestral sequence reconstruction; crystallography; enzymology; epistasis; malate and lactate dehydrogenase; protein evolution; protein stability; trichomonad.

Figures

Figure 1
Figure 1
(A) NADH‐dependent reduction of 2‐ketoacids, the reaction catalyzed by L/MDHs. (B) 2‐ketoacid substrates. Oxaloacetate (blue R group) is reduced in MDHs, Pyruvate (red) is reduced in LDHs, and 2‐ketocaproate (yellow) and 2‐ketoisocaproate (green) are other 2‐ketoacids that may potentially be reduced by L/MDHs. (C) Active site of cytosolic MDHs, LDHs have a different residue at position 91: Gln in canonical LDHs, Lys in Plasmodium LDHs, Gly in Cryptosporidium LDHs, and Leu in trichomonad LDHs.
Figure 2
Figure 2
Phylogeny of Trichomonad LDHs and MDHs estimated by the program BAli‐Phy. Full phylogeny is in Figure S2a. Exterior branches are colored by Genbank notation, where MDH is blue and LDH is red. Interior branches are colored by consensus of all descended branches. The nodes M1, L1, L2, and L3 have posterior probability greater than 99%; see Supporting Information Table S6.
Figure 3
Figure 3
log(k cat/K M), relative to 1 M−1 s−1, for the NADH‐dependent reduction of 2‐ketoacid substrates by ancestral and modern trichomonad L/MDHs. Substrates tested were oxaloacetate (blue), pyruvate (red), 2‐ketocaprotate (yellow), and 2‐ketoisocaproate (green). Enzymes are arranged according to an approximate phylogeny (see Fig. 2 and Supporting Information Fig. S2). M1, M2, and L1 activities are averages of the activities of the PM/MB and BP reconstructions (see Supporting Information Fig. S3). Errors for each enzyme/substrate pair are the largest of the standard error of averaging log(k cat/K M) from two separate experiments or the individual standard errors from curve fitting either set of experimental data.
Figure 4
Figure 4
Comparison of active sites of (A) M1 (slate) and (B) closed Tv LDH (salmon).
Figure 5
Figure 5
Superpositions of trichomonad MDH and LDH structures (top) and RMSD by residue (bottom). (A, B) Superpositions of the open (magenta) and closed (salmon) Tv LDH, Tv MDH 1 (cyan), and M1 (slate) dimers, superimposing chain A only and leaving chain B free. Chain A is displayed in (A) and chain B is displayed in (B). Numbers in the superposition in (A) correspond to numbered peaks in the RMSD versus residue plot. (C, D) Superpositions (A, B) with open Tv LDH removed and RMSDs recalculated.
Figure 6
Figure 6
(A) Substitutions between M1 and inhibitor‐bound or apo Tv LDH (magenta spheres) at either of their dimer interfaces (mapped onto M1 subunit A, grey spheres). (B) Substitutions between M1 and Tv MDH (magenta spheres) at either of their dimer interfaces (mapped onto M1 subunit A, gray spheres). (C) Differences between M1 and inhibitor‐bound Tv LDH (magenta spheres) at the M1 NADH binding site (gray spheres; NADH as green, blue, red, and white sticks).
Figure 7
Figure 7
log(k cat/K M), relative to 1M −1 s−1, for the NADH‐dependent reduction of 2‐ketoacid substrates of ancestral and modern enzyme mutants. Errors are calculated as in Figure 3. (A) Results of the L/R swap at position 91 in ancestral and modern trichomonad enzymes. M1 and L1 (PM/MB reconstructions) data are also in Figure S3; Tv MDH and LDH data are also in Figure 3. (B) Results of other mutations to M1. Locations of the residues mutated in the M1 structure are indicated above the kinetics results, along with the key active site residues H186 and R161, NADH, and phosphate (white).
Figure 8
Figure 8
M1 arginine 91, with distances between the γ‐carbon and other nearby nonhydrogen atoms.

Similar articles

See all similar articles

Cited by 7 articles

See all "Cited by" articles

References

    1. Ohno S. 1970. Evolution by gene duplication. Springer‐Verlag.
    1. Conant GC, Wolfe KH (2008) Turning a hobby into a job: how duplicated genes find new functions. Nature Rev Genet 9:938–950. - PubMed
    1. Innan H, Kondrashov F (2010) The evolution of gene duplications: classifying and distinguishing between models. Nat Rev Genet 11:97–108. - PubMed
    1. Bergthorsson U, Andersson DI, Roth JR (2007) Ohno's dilemma: evolution of new genes under continuous selection. Proc Natl Acad Sci USA 104:17004–17009. - PMC - PubMed
    1. Jensen RA (1976) Enzyme recruitment in evolution of new function. Ann Rev Microbiol 30:409–425. - PubMed

Publication types

LinkOut - more resources

Feedback