An atomic-resolution view of neofunctionalization in the evolution of apicomplexan lactate dehydrogenases

Abstract

Malate and lactate dehydrogenases (MDH and LDH) are homologous, core metabolic enzymes that share a fold and catalytic mechanism yet possess strict specificity for their substrates. In the Apicomplexa, convergent evolution of an unusual LDH from MDH produced a difference in specificity exceeding 12 orders of magnitude. The mechanisms responsible for this extraordinary functional shift are currently unknown. Using ancestral protein resurrection, we find that specificity evolved in apicomplexan LDHs by classic neofunctionalization characterized by long-range epistasis, a promiscuous intermediate, and few gain-of-function mutations of large effect. In canonical MDHs and LDHs, a single residue in the active-site loop governs substrate specificity: Arg102 in MDHs and Gln102 in LDHs. During the evolution of the apicomplexan LDH, however, specificity switched via an insertion that shifted the position and identity of this ‘specificity residue’ to Trp107f. Residues far from the active site also determine specificity, as shown by the crystal structures of three ancestral proteins bracketing the key duplication event. This work provides an unprecedented atomic-resolution view of evolutionary trajectories creating a nascent enzymatic function.DOI: http://dx.doi.org/10.7554/eLife.02304.001

Functional and Structural Resilience of the Active Site Loop in the Evolution of Plasmodium Lactate Dehydrogenase

“…The alanine scan surprisingly revealed that all residues except W107fA tolerate mutation with little to no effect on their catalytic efficiency [ k cat / K M (Figure 3B), as reported previously 3 ]. W107fA has a 4 order of magnitude drop in k cat , indicating that W107f is essential for wild-type levels of strong catalysis.…”

“…3 The structure of W107fA is nearly identical to that of the wild-type protein [root-mean-square deviation of 0.95 Å 2 (Figure 4A)]. The only significant difference between W107A and the wild type is in the orientation of the specificity loop.…”

“…3 The boundaries of the scan were determined from a superposition of the human lactate dehydrogenase (LDH) and Pf LDH. The main chains of the specificity loops in the two structures conformationally diverge between positions 101 and 108, a span of 12 residues.…”

“…Rather, apicomplexan LDHs evolved from an ancestral MDH via a unique five-amino acid insertion in the substrate specificity loop that switches substrate specificity from malate/oxaloacetate to lactate/pyruvate. 3 The insertion lengthens the loop, but otherwise, the structures of Pf LDH and canonical LDHs and MDHs are highly similar (Figure 2A). …”

“…Using point mutations and alanine scanning of the substrate specificity loop, we previously showed that W107f is essential for Pf LDH enzyme activity and substrate recognition, while mutations of K102 have a negligible effect. 3 …”