Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

Babková, Petra; Šebestová, Eva; Brezovský, Jan; Chaloupková, Radka; Damborský, Jiřı́

doi:10.1002/cbic.201700197

Cited by 58 publications

(58 citation statements)

References 103 publications

(171 reference statements)

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“…Overall, our data agree with previous studies showing that the pool of possible ancestors at the nodes of a phylogenetic tree reflect ancestral mechanistic function experimentally [75–77]. In addition, several studies also suggest that proteins have changed from lower to higher specificity over long evolutionary time [78,79].…”

Section: Discussionsupporting

confidence: 91%

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Grinshpon

Shrestha

Titus-McQuillan

et al. 2019

Biochemical Journal

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Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases prefer an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared with caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates a preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.

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Section: Discussionsupporting

confidence: 91%

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Grinshpon

Shrestha

Titus-McQuillan

et al. 2019

Biochemical Journal

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“…Within recent literature, marginal ancestral protein reconstruction has been shown to introduce novel functional properties into proteins 37,46,47 . As ancestral proteins typically trend towards increased stability when sampling from more ancient nodes 40 , we reconstructed the most recent common ancestor of the Nocardia, Streptomyces, and Mycobacterial CARs.…”

Section: Resultsmentioning

confidence: 99%

“…Consequently, a series of studies have used evolutionary histories to isolate sites of interest to engineer enzymes with novel functionality 30–34 . When used as an engineering tool, ASR has produced enzymes with improved stability 35 , substrate ranges 36 , or both 37 . ASR differs from other engineering methods as it generates new sequences based upon probabilistic searches of non-conserved functional space, giving each output a high likelihood of being functional given an accurate sequence alignment input.…”

Section: Introductionmentioning

confidence: 99%

Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction

et al. 2019

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Carboxylic acid reductases (CARs) are biocatalysts of industrial importance. Their properties, especially their poor stability, render them sub-optimal for use in a bioindustrial pipeline. Here, we employed ancestral sequence reconstruction (ASR) – a burgeoning engineering tool that can identify stabilizing but enzymatically neutral mutations throughout a protein. We used a three-algorithm approach to reconstruct functional ancestors of the Mycobacterial and Nocardial CAR1 orthologues. Ancestral CARs (AncCARs) were confirmed to be CAR enzymes with a preference for aromatic carboxylic acids. Ancestors also showed varied tolerances to solvents, pH and in vivo-like salt concentrations. Compared to well-studied extant CARs, AncCARs had a Tm up to 35 °C higher, with half-lives up to nine times longer than the greatest previously observed. Using ancestral reconstruction we have expanded the existing CAR toolbox with three new thermostable CAR enzymes, providing access to the high temperature biosynthesis of aldehydes to drive new applications in biocatalysis.

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“…The ancestral sequence was reconstructed as previously described. 41 Briefly, a nonredundant set of HLD-II protein sequences was collected by PSI-BLAST v2.2.22+ 42 searches against the NCBI nr database (version 21-9-2012) 43 and then clustered with CLANS 44 and CD-HIT v4.5.4. 45 The data set was divided into two subgroups based on their evolutionary relatedness.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…We have recently inferred the sequences of five highly stable ancestral enzymes corresponding to the different nodes of the HLD-II phylogenetic tree. 41 Here, we report construction and detailed biochemical characterization of the common ancestor of HLDs and RLuc, denoted AncHLD-RLuc. The ancestral state distributions at each site of the AncHLD-RLuc node were predicted by the maximum likelihood method using 93 sequences available for the HLD-II subfamily ( Figure S2).…”

Section: Catalytic Promiscuity Of the Reconstructed Common Ancestor Omentioning

confidence: 99%

Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts

et al. 2019

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To obtain structural insights into the emergence of biological functions from catalytically promiscuous enzymes, we reconstructed an ancestor of catalytically distinct, but evolutionarily related, haloalkane dehalogenases (EC 3.8.1.5) and Renilla luciferase (EC 1.13.12.5). This ancestor has both hydrolase and monooxygenase activities. Its crystal structure solved to 1.39 Å resolution revealed the presence of a catalytic pentad conserved in both dehalogenase and luciferase descendants and a molecular oxygen bound in between two residues typically stabilizing a halogen anion. The differences in the conformational dynamics of the specificity-determining cap domains between the ancestral and descendant enzymes were accessed by molecular dynamics and hydrogen−deuterium exchange mass spectrometry. Stopped-flow analysis revealed that the alkyl enzyme intermediate formed in the luciferase-catalyzed reaction is trapped by blockage of a hydrolytic reaction step. A single-point mutation (Ala54Pro) adjacent to one of the catalytic residues bestowed hydrolase activity on the modern luciferase by enabling cleavage of this intermediate. Thus, a single substitution next to the catalytic pentad may enable the emergence of promiscuous activity at the enzyme class level, and ancestral reconstruction has a clear potential for obtaining multifunctional catalysts.

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Ancestral Haloalkane Dehalogenases Show Robustness and Unique Substrate Specificity

Cited by 58 publications

References 103 publications

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity

Highly thermostable carboxylic acid reductases generated by ancestral sequence reconstruction

Light-Emitting Dehalogenases: Reconstruction of Multifunctional Biocatalysts

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