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

Boucher JI, Jacobowitz JR, Beckett BC, Classen S, Theobald DL - Elife (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Brandeis University, Waltham, United States.

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.

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Fold architecture in the LDH and MDH superfamily.At left is CpMDH (blue, 2hjr), at right is PfLDH (vermilion and olive, 1t2d). The Rossmann fold domain, which binds the NADH cofactor, is show as light blue in CpMDH and vermilion in PfLDH. The active site is found at the interface of the two domains. In CpMDH, the ‘opposing loop’ is highlighted in yellow (see text). In PfLDH, the six-residue insertion is highlighted in yellow.DOI:http://dx.doi.org/10.7554/eLife.02304.004
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fig1s1: Fold architecture in the LDH and MDH superfamily.At left is CpMDH (blue, 2hjr), at right is PfLDH (vermilion and olive, 1t2d). The Rossmann fold domain, which binds the NADH cofactor, is show as light blue in CpMDH and vermilion in PfLDH. The active site is found at the interface of the two domains. In CpMDH, the ‘opposing loop’ is highlighted in yellow (see text). In PfLDH, the six-residue insertion is highlighted in yellow.DOI:http://dx.doi.org/10.7554/eLife.02304.004

Mentions: LDH and MDH are homologous, 2-ketoacid oxidoreductases that share both a protein fold (Rossmann et al., 1975; Figure 1—figure supplement 1) and a common catalytic mechanism (Birktoft and Banaszak, 1983; Clarke et al., 1986; Hart et al., 1987a, 1987b; Clarke et al., 1988; Waldman et al., 1988; Figure 1). Both enzymes are found in central metabolism: MDH catalyzes the interconversion of oxaloacetate and malate in the citric acid cycle, and LDH converts pyruvate to lactate in the final step of anaerobic glycolysis. Despite their structural and catalytic similarities, modern apicomplexan LDHs and MDHs have extraordinarily strict substrate specificity. For example, Plasmodium falciparum (Pf) MDH and LDH each prefer their respective substrates by over six orders of magnitude. The biophysical basis for this extraordinary substrate preference is presently an unresolved question.10.7554/eLife.02304.003Figure 1.Schematic of M/LDH superfamily active site and catalytic mechanism.


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

Boucher JI, Jacobowitz JR, Beckett BC, Classen S, Theobald DL - Elife (2014)

Fold architecture in the LDH and MDH superfamily.At left is CpMDH (blue, 2hjr), at right is PfLDH (vermilion and olive, 1t2d). The Rossmann fold domain, which binds the NADH cofactor, is show as light blue in CpMDH and vermilion in PfLDH. The active site is found at the interface of the two domains. In CpMDH, the ‘opposing loop’ is highlighted in yellow (see text). In PfLDH, the six-residue insertion is highlighted in yellow.DOI:http://dx.doi.org/10.7554/eLife.02304.004
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109310&req=5

fig1s1: Fold architecture in the LDH and MDH superfamily.At left is CpMDH (blue, 2hjr), at right is PfLDH (vermilion and olive, 1t2d). The Rossmann fold domain, which binds the NADH cofactor, is show as light blue in CpMDH and vermilion in PfLDH. The active site is found at the interface of the two domains. In CpMDH, the ‘opposing loop’ is highlighted in yellow (see text). In PfLDH, the six-residue insertion is highlighted in yellow.DOI:http://dx.doi.org/10.7554/eLife.02304.004
Mentions: LDH and MDH are homologous, 2-ketoacid oxidoreductases that share both a protein fold (Rossmann et al., 1975; Figure 1—figure supplement 1) and a common catalytic mechanism (Birktoft and Banaszak, 1983; Clarke et al., 1986; Hart et al., 1987a, 1987b; Clarke et al., 1988; Waldman et al., 1988; Figure 1). Both enzymes are found in central metabolism: MDH catalyzes the interconversion of oxaloacetate and malate in the citric acid cycle, and LDH converts pyruvate to lactate in the final step of anaerobic glycolysis. Despite their structural and catalytic similarities, modern apicomplexan LDHs and MDHs have extraordinarily strict substrate specificity. For example, Plasmodium falciparum (Pf) MDH and LDH each prefer their respective substrates by over six orders of magnitude. The biophysical basis for this extraordinary substrate preference is presently an unresolved question.10.7554/eLife.02304.003Figure 1.Schematic of M/LDH superfamily active site and catalytic mechanism.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Brandeis University, Waltham, United States.

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.

Show MeSH
Related in: MedlinePlus