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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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Related in: MedlinePlus

Alternative LDH mutations in AncMDH2.Relative specificity (RS) is described in legend of Figure 4.DOI:http://dx.doi.org/10.7554/eLife.02304.03110.7554/eLife.02304.032Figure 8—source data 1.Kinetic parameters for specificity residue mutants.DOI:http://dx.doi.org/10.7554/eLife.02304.032
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fig8: Alternative LDH mutations in AncMDH2.Relative specificity (RS) is described in legend of Figure 4.DOI:http://dx.doi.org/10.7554/eLife.02304.03110.7554/eLife.02304.032Figure 8—source data 1.Kinetic parameters for specificity residue mutants.DOI:http://dx.doi.org/10.7554/eLife.02304.032

Mentions: Given the known importance of position 102, the ‘specificity residue’, in substrate recognition, we wondered whether different residues at position 102 could confer pyruvate activity. Position 102 in fact differs in the four convergent LDH families: Gln in canonical LDHs (Wilks et al., 1988), Lys in the apicomplexan LDHs, Gly in Cryptosporidium LDHs (Madern et al., 2004), and Leu in trichomonad LDHs (Wu and Fiser, 1999). Could the ancestral apicomplexan MDH have evolved pyruvate specificity by any of these alternative routes? To answer this question, we evaluated the potential of these different amino acids at the 102 position to confer pyruvate specificity in the AncMDH2 background. Each mutation increases pyruvate activity, but none result in a highly specific LDH. The canonical mutation (Arg102Gln) results in the largest gain in pyruvate activity (2800-fold) and the smallest loss of oxaloacetate activity (2500-fold) (Figure 8). Additionally, we tested whether the full six amino acid insertion was required to confer pyruvate specificity in AncMDH2 or if simply mutating Arg102 to Trp was sufficient. The Arg102Trp mutation all but abolishes activity towards both substrates, indicating that the loop insertion was necessary to switch the specificity residue (Figure 8).10.7554/eLife.02304.031Figure 8.Alternative LDH mutations in AncMDH2.


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)

Alternative LDH mutations in AncMDH2.Relative specificity (RS) is described in legend of Figure 4.DOI:http://dx.doi.org/10.7554/eLife.02304.03110.7554/eLife.02304.032Figure 8—source data 1.Kinetic parameters for specificity residue mutants.DOI:http://dx.doi.org/10.7554/eLife.02304.032
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: Alternative LDH mutations in AncMDH2.Relative specificity (RS) is described in legend of Figure 4.DOI:http://dx.doi.org/10.7554/eLife.02304.03110.7554/eLife.02304.032Figure 8—source data 1.Kinetic parameters for specificity residue mutants.DOI:http://dx.doi.org/10.7554/eLife.02304.032
Mentions: Given the known importance of position 102, the ‘specificity residue’, in substrate recognition, we wondered whether different residues at position 102 could confer pyruvate activity. Position 102 in fact differs in the four convergent LDH families: Gln in canonical LDHs (Wilks et al., 1988), Lys in the apicomplexan LDHs, Gly in Cryptosporidium LDHs (Madern et al., 2004), and Leu in trichomonad LDHs (Wu and Fiser, 1999). Could the ancestral apicomplexan MDH have evolved pyruvate specificity by any of these alternative routes? To answer this question, we evaluated the potential of these different amino acids at the 102 position to confer pyruvate specificity in the AncMDH2 background. Each mutation increases pyruvate activity, but none result in a highly specific LDH. The canonical mutation (Arg102Gln) results in the largest gain in pyruvate activity (2800-fold) and the smallest loss of oxaloacetate activity (2500-fold) (Figure 8). Additionally, we tested whether the full six amino acid insertion was required to confer pyruvate specificity in AncMDH2 or if simply mutating Arg102 to Trp was sufficient. The Arg102Trp mutation all but abolishes activity towards both substrates, indicating that the loop insertion was necessary to switch the specificity residue (Figure 8).10.7554/eLife.02304.031Figure 8.Alternative LDH mutations in AncMDH2.

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