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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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Sequence alignment of the specificity loop from apicomplexan M/LDHs with ancestral sequences.DOI:http://dx.doi.org/10.7554/eLife.02304.006
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fig2s1: Sequence alignment of the specificity loop from apicomplexan M/LDHs with ancestral sequences.DOI:http://dx.doi.org/10.7554/eLife.02304.006

Mentions: In contrast, the large apicomplexan LDH clade is demarcated by a unique, conserved five-residue insertion in the active site loop. While the apicomplexan LDH and MDH proteins are moderately divergent, with about 45% sequence identity, the differences are largely confined to exterior residues removed from the active sites. One important difference is that the apicomplexan LDHs have Lys102 for the ‘specificity residue’, rather than a Gln as found in the canonical LDHs (Figure 2—figure supplement 1). Apicomplexan proteins frequently contain numerous insertions relative to proteins from other species (Feng et al., 2006; Kissinger and DeBarry, 2011), a characteristic thought to result from various factors, including high AT genome content, DNA strand slippage, double strand break repair, high recombination rates, and selection pressure for parasite antigenic variation. Except for Met106, the amino acid and coding sequence immediately flanking the apicomplexan LDH loop insertion is largely conserved with α-proteobacterial MDHs (Figure 2—figure supplement 1). It is therefore likely that a mutation ‘expanded’ the Met106 codon to code for six residues, resulting in the observed five-residue insertion and the Met106Lys mutation. Henceforth we will refer to this expansion mutation as the ‘six-residue loop insertion’.


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)

Sequence alignment of the specificity loop from apicomplexan M/LDHs with ancestral sequences.DOI:http://dx.doi.org/10.7554/eLife.02304.006
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2s1: Sequence alignment of the specificity loop from apicomplexan M/LDHs with ancestral sequences.DOI:http://dx.doi.org/10.7554/eLife.02304.006
Mentions: In contrast, the large apicomplexan LDH clade is demarcated by a unique, conserved five-residue insertion in the active site loop. While the apicomplexan LDH and MDH proteins are moderately divergent, with about 45% sequence identity, the differences are largely confined to exterior residues removed from the active sites. One important difference is that the apicomplexan LDHs have Lys102 for the ‘specificity residue’, rather than a Gln as found in the canonical LDHs (Figure 2—figure supplement 1). Apicomplexan proteins frequently contain numerous insertions relative to proteins from other species (Feng et al., 2006; Kissinger and DeBarry, 2011), a characteristic thought to result from various factors, including high AT genome content, DNA strand slippage, double strand break repair, high recombination rates, and selection pressure for parasite antigenic variation. Except for Met106, the amino acid and coding sequence immediately flanking the apicomplexan LDH loop insertion is largely conserved with α-proteobacterial MDHs (Figure 2—figure supplement 1). It is therefore likely that a mutation ‘expanded’ the Met106 codon to code for six residues, resulting in the observed five-residue insertion and the Met106Lys mutation. Henceforth we will refer to this expansion mutation as the ‘six-residue loop insertion’.

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