Gradual neofunctionalization in the convergent evolution of trichomonad lactate and malate dehydrogenases

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.

Robustness of Reconstructed Ancestral Protein Functions to Statistical Uncertainty

“…Previous theoretical and simulation studies showed that incorporating stochastic uncertainty about the underlying phylogeny does not strongly affect ancestral sequence reconstruction ( Hanson-Smith et al 2010 ). Consistent with this finding, several empirical case studies have found that assuming different plausible phylogenies has only weak effects on inferences of ancestral protein functions ( Gaucher et al 2003 ; Akanuma et al 2015 ; Clifton and Jackson 2016 ; Steindel et al 2016 ) Systematic error in the tree, however, could still affect ancestral sequences and their functions ( Groussin et al 2015 ). Further, comprehensive work has not been conducted on the effects of the assumed model on ancestral reconstructions; however, research to date suggests that functional inferences are generally robust to sequence uncertainty associated with using different models and methods ( Ugalde et al 2004 ; Thomson et al 2005 ; Chang et al 2007 ; Devamani et al 2016 ; Steindel et al 2016 ).…”

“…Ancestral protein reconstruction (APR)—phylogenetic inference of ancient protein sequences, followed by gene synthesis, expression, and experimental characterization—has become a widely used strategy to experimentally test hypotheses about the functional and biochemical properties of ancient proteins ( Jermann et al 1995 ; Chandrasekharan et al 1996 ; Chang et al 2002 ; Thornton et al 2003 ; Thomson et al 2005 ; Gaucher et al 2008 ; Hobbs et al 2012 ; Akanuma et al 2013 ; Bar-Rogovsky et al 2013 ; Risso et al 2013 ; Williams et al 2013 ; Boucher et al 2014 ; Akanuma et al 2015 ; Bickelmann et al 2015 ; Carrigan et al 2015 ; Clifton and Jackson 2016 ; Devamani et al 2016 ; Steindel et al 2016 ). APR has also been used to experimentally determine the effects of specific historical changes in protein sequence on the properties of ancient proteins by introducing mutations that recapitulate ancient sequence substitutions into reconstructed ancestral proteins ( Zhang and Rosenberg 2002 ; Bridgham et al 2006 ; Kaiser et al 2007 ; Ortlund et al 2007 ; Yokoyama et al 2008 ; Lynch et al 2011 ; Finnigan et al 2012 ; Harms et al 2013 ; Smith et al 2013 ; Wilson et al 2015 ).…”

Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains

“…The 2-hydroxy-acid dehydrogenase(s) responsible for the reduction of the 2-keto acids to form lactates has not been identified. In lactic acid bacteria (28) and protozoa, such as Trichomonas vaginalis (29), these reactions are catalyzed by the canonical NAD-dependent hydroxy acid dehydrogenase lactate dehydrogenase (LDH), an enzyme with broad substrate specificity (30). Leishmania species do not encode LDH, but genes for malate dehydrogenases (MDH) and a putative D-lactate dehydrogenase (D-LDH) are present.…”

How enzyme promiscuity and horizontal gene transfer contribute to metabolic innovation

“…In this lineage, the common ancestor of LDH and MDH was specific for malate. LDH activity emerged after gene duplication and increased over time, yielding the broad‐specificity Trichomonas vaginalis LDH, which has poor discrimination among pyruvate, lactate, and other 2‐ketoacids .…”

“…Consequently, additional criteria need to be used to determine which model best fits the evolution of a given enzyme. Many studies use ancestral reconstruction to evaluate changes in activity between ancestral enzymes and their descendants . For example, the neofunctionalization model predicts that the ancestor cannot catalyze the activity of the newly evolved enzyme.…”