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. 2022 Nov 13;62(4):651–657. doi: 10.1007/s12088-022-01043-8

Phylogenetic Analyses of Microbial Hydrolytic Dehalogenases Reveal Polyphyletic Origin

Devi Lal 1, Himani Pandey 2, Rup Lal 3,4,✉
PMCID: PMC9705686  PMID: 36458228

Abstract

Hydrolytic dehalogenases form an important class of dehalogenases that include haloacid dehalogenase, haloalkane dehalogenase, haloacetate dehalogenase, and atrazine chlorohydrolase. These enzymes are involved in biodegradation of various environmental pollutants and therefore it is important to understand their phylogeny. In the present study, it was found that the enzymes haloalkane and haloacetate dehalogenases share a common ancestry with enzymes such as carboxyesterase, epoxide hydrolase, and lipases, which can be traced to ancestral α/β hydrolase fold enzyme. Haloacid dehalogenases and atrazine chlorohydrolases have probabaly evolved from ancestral enzymes with phosphatase and deaminases activity, respectively. These findings were supported by the similarities in the secondary structure, key catalytic motifs and placement of catalytic residues. The phylogeny of haloalkane dehalogenases and haloacid dehalogenases differs from 16S rRNA gene phylogeny, suggesting spread through horizontal gene transfer. Hydrolytic dehalogenases are polyphyletic and do not share a common evolutionay history, the functional similarities are due to convergent evolution. The present study also identifies key functional residues, mutating which, can help in generating better enzymes for clean up of the persistent environmental pollutants using enzymatic bioremediation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-022-01043-8.

Keywords: Hydrolytic dehalogenases, Secondary structure, Functional divergence, Convergent evolution, Motifs

Introduction

Halogenated compounds that have been used widely as pesticides and herbicides are hazardous and toxic environmental pollutants [1]. Microbes have been isolated that can degrade these toxic compounds efficiently [2]. Dehalogenases are key enzymes found in such microbes that cleave C-halogen bond by employing various mechanisms [3]. Dehalogenases have been classified based on substrate specificities, reaction kinetics, molecular mechanism, sensitivities to inhibitory compounds, and sequence information. Hydrolytic dehalogenase is an important class of dehalogenases that includes haloacid dehalogenase, haloalkane dehalogenase, haloacetate dehalogenase, and lesser known atrazine chlorohydrolase.

2-haloacid dehalogenases (EC 3.8.1.2) act on 2-haloalkanoic acids to produce corresponding 2-hydroxyalkanoic acids [4]. Haloalkane dehalogenases (EC 3.8.1.5) cleave carbon-halogen bonds of halogenated aliphatic compounds using a water molecule, yielding a primary alcohol, a proton, and a halide [5]. They are capable of converting a number of substrates, such as halogenated alkanes, cycloalkanes, alkenes, ethers, alcohols, ketones, and cyclic dienes. Haloacetate dehalogenases (EC 3.8.1.3) catalyze hydrolysis of haloacetates to produce glycolate. Atrazine chlorohydrolases (EC 3.8.1.8) are the enzymes that initiate degradation of herbicide atrazine used to control weeds [6].

Since all hydrolytic dehalogenases share similarity in function [4–6], it is important to know whether they are evolutionarily related or not. This study presents detailed insights into the phylogeny and evolutionary relatedness of hydrolytic dehalogenases.

Methods

Proteins with hydrolytic dehalogenase activity were screened in KEGG database (Kyoto Encyclopedia of Genes and Genomes). Phyre (Protein Homology/ analogY Recognition Engine) version 2.0 [7] was used for homology search. The sequences of the identified homologous proteins were retrieved from KEGG and SWISS-PROT databases. The sequences were clustered using CLANS (CLuster ANalysis of Sequences) software [8]. The input file for CLANS was generated using an E-value cut off ‘10’ and P value cut off ‘0.1’. Multiple sequence alignments were performed using Clustal_X. Phylogenetic analyses were performed using MEGA11 using neighbor-joining method. Phylogenetic trees of haloacetate dehalogenases and atrazine chlorohydrolases were not inferred due to the small number of sequences available for these two dehalogenases.

For secondary structure prediction, PSIPRED [9], Predict Protein server [10] and Jpred4 [11] were used. Amino acid conservation was obtained from the CONSURF server [12] using WAG model of evolution. Solvent accessibility of individual residue was predicted using Predict Protein server and CONSURF server. Homology modeling was carried out using (PS)2 server [13] and SWISS-MODEL [14]. The structure obtained was analyzed using ProFunc [15]. The volume of active site cavity was calculated using CASTp 3.0 server [16]. MEME suite [17] was used to find the conserved motifs.

Results and Discussion

Homology Prediction

It was found that haloalkane and haloacetate dehalogenases are closely related, belonging to abhydrolase-domain containing proteins (Table 1). Both showed homology with carboxyesterases, epoxide hydrolases, and lipases/esterases and were found to structurally belong to the α/β hydrolase fold family [18]. Haloacid dehalogenases showed similarity with phosphoglycolate phosphatases and other phosphatases, like histidine biosynthesis bifunctional protein. Haloacid dehalogenases could be related to haloalkane and haloacetate dehalogenases due to their similarity with epoxide hydrolases. Atrazine chlorohydrolases were found to be related to amidohydrolases, like guanosine and cytosine deaminases. However, a small dataset is available for atrazine chlorohydrolases and it is likely that as more genomes are sequenced, more such enzymes will be identified.

Table 1.

Homologs identified for different hydrolytic dehalogenases

S. No. Protein Pfam domain Homologs
1. Haloalkane dehalogenases Abhydrolase 1 α/β hydrolase fold, Carboxyesterase, Epoxide hydrolase, Lipase/esterase
2. Haloacetate dehalogenases Abhydrolase 1 Haloalkane dehalogenase, α /β hydrolase fold, Carboxyesterase, Epoxide hydrolase, Lipase/esterase
3. Haloacid dehalogenases Hydrolase Phosphoglycolate phosphatase, Histidine biosynthesis bifunctional protein, Epoxide hydrolase
4. Atrazine chlorohydrolases Amidohydrolase Guanosine deaminase, Cytosine deaminase, Adenosine deaminase
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Cluster Analysis

Haloalkane dehalogenases were found to cluster with haloacetate dehalogenases and other related proteins, like bromoperoxidases, heptadienoate hydrolases, dienelactone hydrolases, and hydroxymuconic semialdehyde hydrolases (Supplementary material Fig. S1). Haloacid dehalogenases were segregated into several smaller groups, suggesting a high sequence diversity. Atrazine chlorohydrolases were found to cluster with other amidohydrolases, like adenosine and cytosine deaminases.

Phylogenetic Analysis

The neighbor-joining tree of haloalkane dehalogenases (Supplementary material Fig. S2) could reveal three clusters or groups. These three groups could also be distinguished based on the placement of the catalytic residues (discussed ahead). The phylogeny obtained for haloalkane dehalogenases was found to differ from 16S rRNA gene-based phylogeny. The grouping of unrelated taxa strongly suggested the role of horizontal gene transfer in acquisition of the genes for haloalkane dehalogenases. The role of HGT in evolution of haloalkane degradation has also been suggested previously [19].

The neighbor-joining tree of haloacid dehalogenases (Supplementary material Fig. S3) could reveal five clusters. Unlike haloalkane dehalogenases, these five clusters could not be differentiated according to the placement of catalytic residues. The phylogeny obtained for haloacid dehalogenases was also found to differ from 16S rRNA gene phylogeny, suggesting the role of horizontal gene transfer in acquisition of these genes.

Sequence Alignment, Catalytic, and Active site Residues

Haloalkane Dehalogenases, Haloacetate Dehalogenases and Related Proteins

The alignment of representatives of haloalkane and haloacetate dehalogenases revealed (Supplementary material Fig. S4):

  • (i)

    The presence of nucleophile Asp (corresponding to the position 108 of LinB from Sphingobium japonicum UT26).

  • (ii)

    A conserved base His (corresponding to the position 272 of LinB from Sphingobium japonicum UT26).

  • (iii)

    Asp or Glu as catalytic acid. Group I and III haloalkane dehalogenases were found to use Asp as catalytic acid (corresponding to the position 250 of DhmA from Mycobacterium avium and DmbB from Mycobacterium tuberculosis H37Rv). Group II haloalkane dehalogenases were found to use Glu/Asp as catalytic acid (corresponding to the position 132 of LinB from Sphingobium japonicum UT26). The catalytic acid in haloacetate dehalogenases was Asp and found to be present at the same position as group II haloalkane dehalogenases. Thus, haloacetate dehalogenases were found to be more closely related to group II haloalkane dehalogenases.

  • (iv)

    A short motif loosely fitting into RVIAPD, corresponding to the position 57–62 of LinB from Sphingobium japonicum UT26, followed by another short motif GxGxS, corresponding to the position 65–69 of LinB from Sphingobium japonicum UT26 (Table 2).

Table 2.

RVIAPD and GxGxS motifs found in haloalkane and haloacetate dehalogenases, and related proteins. The numbers in parentheses indicate the corresponding positions

S. No. Protein Organism RVIAPD motif GxGxS motif
1. Haloalkane Dehalogenase (DhaA) Mycobacterium avium 104 (75) RVVCPD (80) (83) GFGRS (87)
2. Haloalkane Dehalogenase (LinB) Sphingobium japonicum UT26 (57) RLIACD (62) (65) GMGDS (69)
3. Haloalkane Dehalogenase (LinB) Sphingobium indicum B90A (58) RLIACD (63) (66) GMGDS (69)
4. Haloacetate Dehalogenase (DehH) Ralstonia solanacearum (70) TVVATD (75) (78) GYGAS (82)
5. Haloacetate Dehalogenase (DehH ) Azoarcus sp. BH72 (68) TVVASD (73) (76) GYGDA (80)
6. Hydroxymuconic semialdehyde hydrolase (Dmpd) Pseudomonas putida (60) RVIAPD (65) (68) GFGYS (72)
7. Hydroxymuconic semialdehyde hydrolase (XylF) Sphingomonas sp. (63) RVIAPD (68) (71) GFGYS (75)
8. Heptadienoate hydrolase Pseudomonas putida (58) RVIAPD (63) (66) GFGFT (70)
9. Dienelactone hydrolase Burkholderia xenovorans (87) LWQAFD (92) (96) GVGDL (100)
10. Dienelactone hydrolase Pseudomonas sp. (87) LWQAFD (92) (96) GVGDL (100)
11. Bromoperoxidase Streptomyces aureofaciens (53) RVITYD (58) (61) GFGQS (65)
12. Carboxypeptidase Hordeum vulgare (132) NVLFLD (137) (141) GVGFS (145)
13. Acetylcholinesterase Homo sapiens (144) PVLVWI (149) (151) GGGFY (155)
14. Acetylcholinesterase Mus musculus (144) PVLVWI (149) (151) GGGFY (155)
15. Soluble epoxide hydrolase Homo sapiens (287) RVLAMD (292) (295) GYGES (299)
16. Soluble epoxide hydrolase Mus musculus (285) RVLAID (290) (293) GYGDS (297)
17. Microsomal epoxide hydrolase Homo sapiens (179) EVICPS (184) (187) GYGFS (191)
18. Microsomal epoxide hydrolase Mus musculus (179) EVICPS (184) (187) GYGFS (191)
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Haloalkane and haloacetate dehalogenases, and related proteins were found to contain a conserved catalytic motif with a highly conserved base His (Supplementary material Fig. S5).

A comparison of the nucleophile motif suggested three groups (Fig. 1). One group included the proteins that use Asp as the nucleophile. This group included haloalkane dehalogenases, haloacetate dehalogenases, and soluble and microsomal epoxide hydrolases. Second group included proteins that use Ser as the nucleophile. This group included heptadienoate hydrolase, bromoperoxidase, carboxypeptidase, lipase, acetylcholinesterase, and hydroxymuconic semialdehyde hydrolase. Third group included proteins that use Cys as the nucleophile. This group included dienelactone hydrolases. Irrespetive of the nucleophile used, the nucleophile elbow was found to fit into the consensus Sm-X-Nu-X-Sm-Sm, where Sm denotes small amino acid (generally glycine), X denotes any amino acid, while Nu denotes the nucleophile.

Fig. 1.

Fig. 1

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Nucleophile motif found in haloalkane and haloacetate dehalogenases, and related proteins. The numbers in parentheses indicate the corresponding positions. Star indicates the position of the nucleophile

Analysis of catalytic acid motif suggested three possible groups (Fig. 2). One group included proteins that use Asp as catalytic acid. This group included haloacetate dehalogenases, bromo peroxidases, carboxypeptidases, dienelactone hydrolases, heptadienoate hydrolases, and hydroxymuconic semialdehyde hydrolases. Second group included proteins that use Glu as catalytic acid. This group included lipases and acetylcholinesterases. Haloalkane dehalogenases were found in both these groups as they use both Asp and Glu as catalytic acid. Third group included proteins that use Tyr as catalytic acid. This group included soluble and microsomal epoxide hydrolases.

Fig. 2.

Fig. 2

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Catalytic acid motif found in haloalkane and haloacetate dehalogenases, and related proteins. The numbers in parentheses indicate the corresponding positions. Star indicates the position of catalytic acid

Haloacid Dehalogenases and Related Proteins

A conserved Asp residue, corresponding to the position 8 of haloacid dehalogenase from Xanthobacter autotrophicus, was found in all haloacid dehalogenases analyzed in the present study (Supplementary material Fig. S6). This conserved Asp acts as the nucleophile. Site-directed mutagenesis on Pseudomonas sp. L-2-haloacid dehalogenase has also shown that Asp10 is essential for enzymatic activity [20]. It can be postulated that Asp residue is evolutionary conserved, despite divergence for different substrates.

It was found that all the proteins related to haloacid dehalogenases contained a conserved motif with a highly conserved Asp residue (Supplementary material Fig. S7). Soluble epoxide hydrolases were also found to contain a similar motif with a highly conserved Asp residue, which is not the nucleophile (soluble epoxide hydrolases use Asp333 nucleophile, corresponding to the position of soluble epoxide hydrolase from Mus musculus). The presence of motif like haloacid dehalogenases with highly conserved Asp, and highly conserved base His and catalytic triad like haloalkane dehalogenases suggested that both haloalkane and haloacid dehalogenases may share an ancestry with soluble epoxide hydrolases. No such motif could be identified in microsomal epoxide hydrolases. It may be postulated that soluble epoxide hydrolases may have evolved by gene fusion of the ancestral haloacid and haloalkane dehalogenase-like proteins.

Atrazine Chlorohydrolases and Related Proteins

A number of conserved residues were identified in atrazine chlorohydrolases and related proteins that may play an important role in catalysis (Supplementary material Fig. S8). Atrazine chlorohydrolases and their homologs have some perfectly conserved amino acid residues, including His66, His68, His243, His276, and Asp327 (all numbers corresponding to the position of AtzA from Pseudomonas sp. ADP). His276 was found to be related to the conserved His residue of adenosine and cytosine deaminases, which forms hydrogen bond with nucleophilic water (Supplementary material Fig. S9a-d).

These residues were found to be involved in metal binding in cytosine deaminase [21] and adenosine deaminase [22]. Thus, metal binding sites present in atrazine chlorohydrolases were also found to be perfectly conserved across cytosine and adenosine deaminases. This suggests that these proteins share a common ancestry and during molecular evolution, they adapted to perform different functions, but the metal binding sites remained perfectly conserved. Atrazine chlorohydrolases were thus found to differ from other hydrolytic dehalogenases as they use metal ions for catalysis.

Secondary Structure Predictions and Position of Catalytic Residues

Haloalkane Dehalogenases, Haloacetate Dehalogenases, and Related Proteins

The predicted secondary structures for haloalkane and haloacetate dehalogenases were similar with eight β sheets (Supplementary material Fig. S10, all predicted structures are not shown). Protein modeling and topology analyses suggested that α helices from 4 to 7 formed the cap domain in haloalkane dehalogenases (Supplementary material Fig. S11a). Secondary structures of bromoperoxidases, heptadienoate hydrolases, dienelactone hydrolases, and hydroxymuconic semialdehyde hydrolases were found to be similar to both haloalkane and haloacetate dehalogenases (Supplementary material Fig. S12-S14) (all predicted structures are not shown). The position of the conserved catalytic residues on the secondary structures were analyzed. All the three groups of haloalkane dehalogenases were found to have the nucleophile Asp on the elbow between β5 and α3 (Supplementary material Fig. S12-S14). The position of conserved base His was also found to be constant, i.e., after β8. This pattern was found in all haloalkane dehalogenases, haloacetate dehalogenases and all related proteins. In group I and III haloalkane dehalogenases, catalytic acid was found to be present after β7 strand, while group II haloalkane dehalogenases were found to have the catalytic acid after β6 strand. It has been proposed that the catalytic acid shifted from β6 to β7 strand in DhlA from Xanthobacter autotrophicus, which is thought to be an important event in the adaptation towards 1,2-dichloroethane [23].

The catalytic acid in bromoperoxidases, heptadienoate hydrolases, dienelactone hydrolases, and hydroxymuconic semialdehyde hydrolases was found to be located after β7 strand, like group I and III haloalkane dehalogenases. Haloacetate dehalogenases were found to have catalytic acid located after β6 strand, suggesting that these are closely related to group II haloalkane dehalogenases. The C-terminus of microsomal and soluble epoxide hydrolases (corresponding to the region 115–445 of microsomal epoxide hydrolase and 220–554 of soluble epoxide hydrolase from Mus musculus) was found to be similar to haloalkane dehalogenases (Supplementary material Fig. S15, S16). This similarity was also reported by Beetham et al. [24]. Moreover, the nucleophile Asp in microsomal and soluble epoxide hydrolases was found to be located in the elbow between β7 and α8, as found in haloalkane dehalogenases.

The presence of a catalytic triad with conserved His base, conserved motifs, and similarity in the secondary structures suggests that haloalkane and haloacetate dehalogenases, along with related proteins, like bromoperoxidase, heptadienoate hydrolase, dienelactone hydrolase, and hydroxymuconic semialdehyde hydrolase share a common ancestry. These proteins diverged to perform different functions, with conserved residues contributing to structural and conformation stability.

Haloacid Dehalogenases and Related Proteins

Secondary structure of haloacid dehalogenases revealed the presence of seven β sheets (Supplementary material Fig. S17a, all predicted structures are not shown). Protein modeling and topology analyses suggested that α helices from 1 to 4 form the cap domain (Supplementary material Fig. S11b), like haloalkane dehalogenases. The structure and topology of haloacid dehalogenases suggested a structure similar to haloalkane and haloacetate dehalogenases. However, the structure of haloacid dehalogenases could be differentiated from latter due to the difference in the position of nucleophile and absence of a catalytic triad. The structure of 2-phosphoglycolate phosphatases was found to be similar to haloacid dehalogenases (Supplementary material Fig. S17, all predicted structures are not shown). The nucleophile Asp was found to be present on the loop, just after the first β strand. The N-terminus of soluble epoxide hydrolases was found to share similarity with the secondary structure of haloacid dehalogenases.

Atrazine Chlorohydrolases and Related Proteins

Atrazine chlorohydrolases contain 17 β sheets (Supplementary material Fig. S18a, all predicted structures are not shown). The secondary structure of atrazine chlorohydrolase AtzA from Pseudomonas sp. ADP was found to resemble the secondary structure of cytosine deaminase from E. coli (Supplementary material Fig. S18a, b). The structure of cytosine deaminase consists of two domains: one formed of (αβ) barrel (position 59–342 of cytosine deaminase from E. coli), which contains the active site and a small β-sandwich domain. The metal binding sites were found to position inside the barrel. The positions of the metal binding sites in AtzA from Pseudomonas sp. were found to be similar to cytosine deaminase from E. coli. Holm and Sander [25] also predicted a structural similarity in the amidohydrolases based on evolutionary relationships.

Volume of Active Site Cavity

The differences in the volume of active sites were found within haloacid dehalogenases, haloalkane dehalogenases, and atrazine chlorohydrolases (Supplementary material Table 1). The differences in the volume of active site cavity of haloacid and haloalkane dehalogenases could be due to a wide range of substrates used by these enzymes. These differences suggested a divergence for different substrates.

Differences in the volume of the active site cavities of atrazine chlorohydrolases were also seen. It is difficult to answer these differences as atrazine chlorohydrolases have only one preferred substrate, i.e., atrazine. Moreover, most of these dehalogenases have been identified by annotation only and have not been experimentally characterized. It is possible that these annotated dehalogenases may not have dehalogenase activity in vivo.

The study provides strong evidence that haloalkane and haloacetate dehalogenases have a common origin, which can be traced to the ancestral α/β fold enzyme, haloacid dehalogenases share ancestry with phosphatases, while atrazine chlorohydrolases have common ancestry with deaminases, like cytosine and adenosine deaminases. Sustained evolutionary pressure resulted in sequence and functional divergence of these enzymes. Therefore, hydrolytic dehalogenases are polyphyletic and the functional similarity is purely because of functional convergence. The present study also identifies key amino acid residues present in different hydrolytic dehlogenases. These residues can be targeted for generating more efficient enzymes for enzymatic bioremediation, which is a newer approach for clean up of the environmental pollutants.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (6.6MB, doc)

Author Contributions

All authors contributed to the study conception and design. Data collection and analysis were performed by Devi Lal, Himani Pandey, and Rup Lal. All authors wrote, read, and approved the final manuscript.

Declarations

Conflict of interest

The authors declare that there are no relevant financial or non-financial competing interests to report.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

Supplementary Material 1 (6.6MB, doc)

Articles from Indian Journal of Microbiology are provided here courtesy of Springer

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