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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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Alternative ancestral enzymes.INS refers to the reconstructed six amino acid insertion from AncLDH*. 58Mut refers to remaining residue differences between AncMDH* and AncLDH* that are not R102K or INS. Relative specificity (RS) is described in legend of Figure 4.DOI:http://dx.doi.org/10.7554/eLife.02304.028
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fig6s7: Alternative ancestral enzymes.INS refers to the reconstructed six amino acid insertion from AncLDH*. 58Mut refers to remaining residue differences between AncMDH* and AncLDH* that are not R102K or INS. Relative specificity (RS) is described in legend of Figure 4.DOI:http://dx.doi.org/10.7554/eLife.02304.028

Mentions: The alternative ancestral reconstructions behave very similar to the prior reconstructions. AncMDH2* is a strict MDH, and AncLDH* is a strict LDH (Figure 6, Figure 6—figure supplement 7). Addition of the six-residue insertion from AncLDH* to AncMDH2* confers pyruvate specificity without adversely affecting oxaloacetate activity (AncMDH2*-INS, Figure 6—figure supplement 7). In the AncMDH2* background, mutating Arg102 to Lys together with the 58 mutations from AncLDH* yields a poor enzyme with little pyruvate activity (AncMDH2*-R102K-58Mut). The kinetic behavior of these AncMDH2* constructs closely matches those seen with the corresponding AncMDH2 constructs (AncMDH2, AncMDH2-INS, and AncMDH2-R102K-59Mut, Figure 6).


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 ancestral enzymes.INS refers to the reconstructed six amino acid insertion from AncLDH*. 58Mut refers to remaining residue differences between AncMDH* and AncLDH* that are not R102K or INS. Relative specificity (RS) is described in legend of Figure 4.DOI:http://dx.doi.org/10.7554/eLife.02304.028
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6s7: Alternative ancestral enzymes.INS refers to the reconstructed six amino acid insertion from AncLDH*. 58Mut refers to remaining residue differences between AncMDH* and AncLDH* that are not R102K or INS. Relative specificity (RS) is described in legend of Figure 4.DOI:http://dx.doi.org/10.7554/eLife.02304.028
Mentions: The alternative ancestral reconstructions behave very similar to the prior reconstructions. AncMDH2* is a strict MDH, and AncLDH* is a strict LDH (Figure 6, Figure 6—figure supplement 7). Addition of the six-residue insertion from AncLDH* to AncMDH2* confers pyruvate specificity without adversely affecting oxaloacetate activity (AncMDH2*-INS, Figure 6—figure supplement 7). In the AncMDH2* background, mutating Arg102 to Lys together with the 58 mutations from AncLDH* yields a poor enzyme with little pyruvate activity (AncMDH2*-R102K-58Mut). The kinetic behavior of these AncMDH2* constructs closely matches those seen with the corresponding AncMDH2 constructs (AncMDH2, AncMDH2-INS, and AncMDH2-R102K-59Mut, Figure 6).

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