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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

Crystal structure of PfLDH-W107fA mutant.(A) Crystal lattice of the W107fA mutant (left) compared to the WT PfLDH (right). (B) Superposition of the WT PfLDH (olive) and the W107fA mutant (vermilion). The structures are highly similar throughout, expect for the active site loop (at top), which is closed in the WT and partially disordered and open in the mutant.DOI:http://dx.doi.org/10.7554/eLife.02304.009
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fig2s3: Crystal structure of PfLDH-W107fA mutant.(A) Crystal lattice of the W107fA mutant (left) compared to the WT PfLDH (right). (B) Superposition of the WT PfLDH (olive) and the W107fA mutant (vermilion). The structures are highly similar throughout, expect for the active site loop (at top), which is closed in the WT and partially disordered and open in the mutant.DOI:http://dx.doi.org/10.7554/eLife.02304.009

Mentions: To assess the effects of Trp107fAla mutation on the specificity loop conformation, we solved the crystal structure of PfLDH-W107fA (1.1 Å ) in the presence of oxamate and NADH. The protein crystallizes in the same space group as the wild-type PfLDH, with nearly identical cell dimensions (Figure 2—figure supplement 3A). In the W107fA mutant, the specificity loop is disordered between residues Thr101 and Arg109, as is often seen in structures in which the loop is in the open conformation. In the mutant, residues 112–115 are in a linear α-helical conformation, in contrast to the wild-type PfLDH closed state which has a very prominent 60° kink in the α-helix at Pro114. Thus, the only significant difference between the wild-type and mutant structures is that the PfLDH-W107fA specificity loop is found in the open conformation, consistent with weaker binding of substrate (Figure 2—figure supplement 3B). These results indicate that Trp107f is necessary for pyruvate activity in apicomplexan LDHs, and that it has become the new ‘specificity residue’ despite the fact that Trp107f does not align in sequence with the canonical specificity residue at position 102 (Figure 2—figure supplement 1).


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)

Crystal structure of PfLDH-W107fA mutant.(A) Crystal lattice of the W107fA mutant (left) compared to the WT PfLDH (right). (B) Superposition of the WT PfLDH (olive) and the W107fA mutant (vermilion). The structures are highly similar throughout, expect for the active site loop (at top), which is closed in the WT and partially disordered and open in the mutant.DOI:http://dx.doi.org/10.7554/eLife.02304.009
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2s3: Crystal structure of PfLDH-W107fA mutant.(A) Crystal lattice of the W107fA mutant (left) compared to the WT PfLDH (right). (B) Superposition of the WT PfLDH (olive) and the W107fA mutant (vermilion). The structures are highly similar throughout, expect for the active site loop (at top), which is closed in the WT and partially disordered and open in the mutant.DOI:http://dx.doi.org/10.7554/eLife.02304.009
Mentions: To assess the effects of Trp107fAla mutation on the specificity loop conformation, we solved the crystal structure of PfLDH-W107fA (1.1 Å ) in the presence of oxamate and NADH. The protein crystallizes in the same space group as the wild-type PfLDH, with nearly identical cell dimensions (Figure 2—figure supplement 3A). In the W107fA mutant, the specificity loop is disordered between residues Thr101 and Arg109, as is often seen in structures in which the loop is in the open conformation. In the mutant, residues 112–115 are in a linear α-helical conformation, in contrast to the wild-type PfLDH closed state which has a very prominent 60° kink in the α-helix at Pro114. Thus, the only significant difference between the wild-type and mutant structures is that the PfLDH-W107fA specificity loop is found in the open conformation, consistent with weaker binding of substrate (Figure 2—figure supplement 3B). These results indicate that Trp107f is necessary for pyruvate activity in apicomplexan LDHs, and that it has become the new ‘specificity residue’ despite the fact that Trp107f does not align in sequence with the canonical specificity residue at position 102 (Figure 2—figure supplement 1).

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