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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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Specificity switching in apicomplexan M/LDHs.Blue horizontal bars (left) quantify activity towards oxaloacetate; vermilion horizontal bars (right) quantify activity towards pyruvate. Activity is measured as log10(kcat/KM ), where kcat/KM is in units of s-1M−1. Error bars are shown as small black brackets and represent 1 SD of the mean from triplicate measurements. INS refers to the presence of the six-residue insertion from PfLDH, DEL refers to the removal of the six-residue insertion. Relative specificity (RS) is the ratio of kcat/KM for pyruvate vs oxaloacetate, with positive log10(RS) representing a preference for pyruvate and negative log10(RS) representing a preference for oxaloacetate. All logarithms are base 10.DOI:http://dx.doi.org/10.7554/eLife.02304.01410.7554/eLife.02304.015Figure 4—source data 1.Kinetic parameters for modern constructs.DOI:http://dx.doi.org/10.7554/eLife.02304.015
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fig4: Specificity switching in apicomplexan M/LDHs.Blue horizontal bars (left) quantify activity towards oxaloacetate; vermilion horizontal bars (right) quantify activity towards pyruvate. Activity is measured as log10(kcat/KM ), where kcat/KM is in units of s-1M−1. Error bars are shown as small black brackets and represent 1 SD of the mean from triplicate measurements. INS refers to the presence of the six-residue insertion from PfLDH, DEL refers to the removal of the six-residue insertion. Relative specificity (RS) is the ratio of kcat/KM for pyruvate vs oxaloacetate, with positive log10(RS) representing a preference for pyruvate and negative log10(RS) representing a preference for oxaloacetate. All logarithms are base 10.DOI:http://dx.doi.org/10.7554/eLife.02304.01410.7554/eLife.02304.015Figure 4—source data 1.Kinetic parameters for modern constructs.DOI:http://dx.doi.org/10.7554/eLife.02304.015

Mentions: During evolution, the six-residue insertion displaced the canonical specificity residue at position 102 and apparently switched substrate preference in apicomplexan LDHs. If this insertion is sufficient for pyruvate recognition, then adding the insertion to a modern apicomplexan MDH should convert the enzyme to an LDH. To test this hypothesis, we incorporated the six-residue insertion from PfLDH into the catalytic loop of PfMDH (PfMDH-INS) and the Cryptosporidium parvum (Cp) MDH (CpMDH-INS). The chimeric proteins showed a >100-fold reduction in oxaloacetate activity with no significant gain in pyruvate activity (Figure 4). Like other MDHs, the apicomplexan MDHs have an Arg at position 102 that is important for oxaloacetate recognition; in the modern apicomplexan LDHs position 102 is a Lys. The Arg102Lys mutation may be necessary to eliminate oxaloacetate activity and increase pyruvate activity. Therefore, we also mutated Arg102 to Lys in the PfMDH chimera (PfMDH-R102K-INS). However, this mutation reduced activity towards oxaloacetate by another 100-fold, with no increase in pyruvate activity (Figure 4).10.7554/eLife.02304.014Figure 4.Specificity switching in apicomplexan M/LDHs.


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)

Specificity switching in apicomplexan M/LDHs.Blue horizontal bars (left) quantify activity towards oxaloacetate; vermilion horizontal bars (right) quantify activity towards pyruvate. Activity is measured as log10(kcat/KM ), where kcat/KM is in units of s-1M−1. Error bars are shown as small black brackets and represent 1 SD of the mean from triplicate measurements. INS refers to the presence of the six-residue insertion from PfLDH, DEL refers to the removal of the six-residue insertion. Relative specificity (RS) is the ratio of kcat/KM for pyruvate vs oxaloacetate, with positive log10(RS) representing a preference for pyruvate and negative log10(RS) representing a preference for oxaloacetate. All logarithms are base 10.DOI:http://dx.doi.org/10.7554/eLife.02304.01410.7554/eLife.02304.015Figure 4—source data 1.Kinetic parameters for modern constructs.DOI:http://dx.doi.org/10.7554/eLife.02304.015
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Specificity switching in apicomplexan M/LDHs.Blue horizontal bars (left) quantify activity towards oxaloacetate; vermilion horizontal bars (right) quantify activity towards pyruvate. Activity is measured as log10(kcat/KM ), where kcat/KM is in units of s-1M−1. Error bars are shown as small black brackets and represent 1 SD of the mean from triplicate measurements. INS refers to the presence of the six-residue insertion from PfLDH, DEL refers to the removal of the six-residue insertion. Relative specificity (RS) is the ratio of kcat/KM for pyruvate vs oxaloacetate, with positive log10(RS) representing a preference for pyruvate and negative log10(RS) representing a preference for oxaloacetate. All logarithms are base 10.DOI:http://dx.doi.org/10.7554/eLife.02304.01410.7554/eLife.02304.015Figure 4—source data 1.Kinetic parameters for modern constructs.DOI:http://dx.doi.org/10.7554/eLife.02304.015
Mentions: During evolution, the six-residue insertion displaced the canonical specificity residue at position 102 and apparently switched substrate preference in apicomplexan LDHs. If this insertion is sufficient for pyruvate recognition, then adding the insertion to a modern apicomplexan MDH should convert the enzyme to an LDH. To test this hypothesis, we incorporated the six-residue insertion from PfLDH into the catalytic loop of PfMDH (PfMDH-INS) and the Cryptosporidium parvum (Cp) MDH (CpMDH-INS). The chimeric proteins showed a >100-fold reduction in oxaloacetate activity with no significant gain in pyruvate activity (Figure 4). Like other MDHs, the apicomplexan MDHs have an Arg at position 102 that is important for oxaloacetate recognition; in the modern apicomplexan LDHs position 102 is a Lys. The Arg102Lys mutation may be necessary to eliminate oxaloacetate activity and increase pyruvate activity. Therefore, we also mutated Arg102 to Lys in the PfMDH chimera (PfMDH-R102K-INS). However, this mutation reduced activity towards oxaloacetate by another 100-fold, with no increase in pyruvate activity (Figure 4).10.7554/eLife.02304.014Figure 4.Specificity switching in apicomplexan M/LDHs.

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