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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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Alanine scanning of PfLDH specificity loop.Logarithm of pyruvate kcat/KM of PfLDH and each mutant. Labels on x-axis describe the mutation tested in the WT PfLDH background.DOI:http://dx.doi.org/10.7554/eLife.02304.00710.7554/eLife.02304.008Figure 2—figure supplement 2—source data 1.Kinetic parameters for PfLDH alanine-scan.DOI:http://dx.doi.org/10.7554/eLife.02304.008
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fig2s2: Alanine scanning of PfLDH specificity loop.Logarithm of pyruvate kcat/KM of PfLDH and each mutant. Labels on x-axis describe the mutation tested in the WT PfLDH background.DOI:http://dx.doi.org/10.7554/eLife.02304.00710.7554/eLife.02304.008Figure 2—figure supplement 2—source data 1.Kinetic parameters for PfLDH alanine-scan.DOI:http://dx.doi.org/10.7554/eLife.02304.008

Mentions: We tested the functional importance of residues in the specificity loop in PfLDH with an ‘alanine scan’ by individually mutating each residue in positions 101–108 to an alanine (Figure 2—figure supplement 2, note Ala103 was mutated to a serine). We assessed the activity of the mutants using kcat/Km, a measure of enzymatic specificity and catalytic efficiency, as determined from steady state kinetic assays. Mutating Trp107f to Ala reduced pyruvate activity by five orders of magnitude, whereas mutations at all other positions had effects less than a single order of magnitude, including the canonical specificity residue at position 102. The Trp107fAla mutation affects both kcat (1500-fold decrease) and Km (50-fold increase).


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)

Alanine scanning of PfLDH specificity loop.Logarithm of pyruvate kcat/KM of PfLDH and each mutant. Labels on x-axis describe the mutation tested in the WT PfLDH background.DOI:http://dx.doi.org/10.7554/eLife.02304.00710.7554/eLife.02304.008Figure 2—figure supplement 2—source data 1.Kinetic parameters for PfLDH alanine-scan.DOI:http://dx.doi.org/10.7554/eLife.02304.008
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2s2: Alanine scanning of PfLDH specificity loop.Logarithm of pyruvate kcat/KM of PfLDH and each mutant. Labels on x-axis describe the mutation tested in the WT PfLDH background.DOI:http://dx.doi.org/10.7554/eLife.02304.00710.7554/eLife.02304.008Figure 2—figure supplement 2—source data 1.Kinetic parameters for PfLDH alanine-scan.DOI:http://dx.doi.org/10.7554/eLife.02304.008
Mentions: We tested the functional importance of residues in the specificity loop in PfLDH with an ‘alanine scan’ by individually mutating each residue in positions 101–108 to an alanine (Figure 2—figure supplement 2, note Ala103 was mutated to a serine). We assessed the activity of the mutants using kcat/Km, a measure of enzymatic specificity and catalytic efficiency, as determined from steady state kinetic assays. Mutating Trp107f to Ala reduced pyruvate activity by five orders of magnitude, whereas mutations at all other positions had effects less than a single order of magnitude, including the canonical specificity residue at position 102. The Trp107fAla mutation affects both kcat (1500-fold decrease) and Km (50-fold increase).

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