Haloacid Dehalogenase Enzymes Research Articles

The Eyes Absent Proteins: Unusual HAD Family Tyrosine Phosphatases

Here, we review the haloacid dehalogenase (HAD) class of protein phosphatases, with a particular emphasis on an unusual group of enzymes, the eyes absent (EYA) family. EYA proteins have the unique distinction of being structurally and mechanistically classified as HAD enzymes, yet, unlike other HAD phosphatases, they are protein tyrosine phosphatases (PTPs). Further, the EYA proteins are unique among the 107 classical PTPs in the human genome because they do not use a Cysteine residue as a nucleophile in the dephosphorylation reaction. We will provide an overview of HAD phosphatase structure-function, describe unique features of the EYA family and their tyrosine phosphatase activity, provide a brief summary of the known substrates and cellular functions of the EYA proteins, and speculate about the evolutionary origins of the EYA family of proteins.

Haloacid Dehalogenase Proteins: Novel Mediators of Metabolic Plasticity in Plasmodium falciparum.

Widespread antimalarial drug resistance has prompted the need for new therapeutics and greater understanding of malaria parasite biology. To this end, the isoprenoid biosynthesis inhibitor fosmidomycin has been used to probe the metabolic regulation in the malaria parasite, Plasmodium falciparum. Genetic changes in the haloacid dehalogenase (HAD) superfamily member HAD2 conferred resistance to fosmidomycin, at the cost of decreased fitness. In the absence of fosmidomycin, parasites gained mutations to phosphofructokinase that restored growth and fosmidomycin sensitivity, thus revealing an intriguing example of plasticity in a core glycolytic process. Moreover, this study marks a second report of a HAD superfamily protein-modulating metabolic homeostasis in P falciparum parasites. Haloacid dehalogenase enzymes are distributed across all domains of life and have increasingly been found to influence central carbon metabolism and drug sensitivity in P falciparum. Investigating the mechanisms by which HAD superfamily members modulate metabolism may shed light on how metabolic networks are connected in apicomplexan parasites and other organisms and may guide future therapeutic endeavors.

Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis.

The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family.

Deletion of PHO13, encoding haloacid dehalogenase type IIA phosphatase, results in upregulation of the pentose phosphate pathway in Saccharomyces cerevisiae.

The haloacid dehalogenase (HAD) superfamily is one of the largest enzyme families, consisting mainly of phosphatases. Although intracellular phosphate plays important roles in many cellular activities, the biological functions of HAD enzymes are largely unknown. Pho13 is 1 of 16 putative HAD enzymes in Saccharomyces cerevisiae. Pho13 has not been studied extensively, but previous studies have identified PHO13 to be a deletion target for the generation of industrially attractive phenotypes, namely, efficient xylose fermentation and high tolerance to fermentation inhibitors. In order to understand the molecular mechanisms underlying the improved xylose-fermenting phenotype produced by deletion of PHO13 (pho13Δ), we investigated the response of S. cerevisiae to pho13Δ at the transcriptomic level when cells were grown on glucose or xylose. Transcriptome sequencing analysis revealed that pho13Δ resulted in upregulation of the pentose phosphate (PP) pathway and NADPH-producing enzymes when cells were grown on glucose or xylose. We also found that the transcriptional changes induced by pho13Δ required the transcription factor Stb5, which is activated specifically under NADPH-limiting conditions. Thus, pho13Δ resulted in the upregulation of the PP pathway and NADPH-producing enzymes as a part of an oxidative stress response mediated by activation of Stb5. Because the PP pathway is the primary pathway for xylose, its upregulation by pho13Δ might explain the improved xylose metabolism. These findings will be useful for understanding the biological function of S. cerevisiae Pho13 and the HAD superfamily enzymes and for developing S. cerevisiae strains with industrially attractive phenotypes.

Molecular evolution of the bacterial pseudouridine-5'-phosphate glycosidase protein family.

Pseudouridine is a noncanonical C-nucleoside commonly present in RNA, which is not metabolized in mammals, but can be recycled by the unique enzyme family of bacterial pseudouridine glycosidases such as YeiN from Escherichia coli. Here, we present rigorous bioinformatic and biochemical analyses of the protein family in order to find sequences that might code for nonpseudouridine glycosidase activities. To date, the only other function reported for the enzyme family occurs during the biosynthesis of the antibiotic alnumycin A in Streptomyces species, where AlnA functions as an unusual C-glycosynthase. Bioinformatics analysis of 755 protein sequences identified one group of sequences that were unlikely to harbour pseudouridine glycosidase activities. This observation was confirmed in vitro with one representative protein, IdgA from Streptomyces albus, which was unable to synthesize pseudouridine monophosphate, but was able to attach d-ribose-5-phosphate to juglone. Furthermore, our analyses provide evidence for horizontal gene transfer of pseudouridine glycosidases that may have occurred in Streptomyces and Doria species. Inspection of the genomic loci in the vicinity of pseudouridine glycosidases revealed that in 77% of the strains a kinase gene putatively involved in the phosphorylation of pseudouridine was found nearby, whereas the sequences encoding nonpseudouridine glycosidases coexisted with a phosphatase of the haloacid dehalogenase enzyme family. The investigation suggested that these unknown sequences might be involved in the biosynthesis of soluble blue pigments because of the presence of genes homologous to nonribosomal peptide synthetases.

Structural Basis for the Divergence of Substrate Specificity and Biological Function within HAD Phosphatases in Lipopolysaccharide and Sialic Acid Biosynthesis

The haloacid dehalogenase enzyme superfamily (HADSF) is largely composed of phosphatases that have been particularly successful at adaptating to novel biological functions relative to members of other phosphatase families. Herein, we examine the structural basis for the divergence of function in two bacterial homologues: 2-keto-3-deoxy-d-manno-octulosonate 8-phosphate phosphohydrolase (KDO8P phosphatase, KDO8PP) and 2-keto-3-deoxy-9-O-phosphonononic acid phosphohydrolase (KDN9P phosphatase, KDN9PP). KDO8PP and KDN9PP catalyze the final step in KDO and KDN synthesis, respectively, prior to transfer to CMP to form the activated sugar nucleotide. KDO8PP and KDN9PP orthologs derived from an evolutionarily diverse collection of bacterial species were subjected to steady-state kinetic analysis to determine their specificities toward catalyzed KDO8P and KDN9P hydrolysis. Although each enzyme was more active with its biological substrate, the degree of selectivity (as defined by the ratio of kcat/Km for KDO8P vs KDN9P) varied significantly. High-resolution X-ray structure determination of Haemophilus influenzae KDO8PP bound to KDO/VO3(-) and Bacteriodes thetaiotaomicron KDN9PP bound to KDN/VO3(-) revealed the substrate-binding residues. The structures of the KDO8PP and KDN9PP orthologs were also determined to reveal the differences in their active-site structures that underlie the variation in substrate preference. Bioinformatic analysis was carried out to define the sequence divergence among KDN9PP and KDO8PP orthologs. The KDN9PP orthologs were found to exist as single-domain proteins or fused with the pathway nucleotidyl transferases; the fusion of KDO8PP with the transferase is rare. The KDO8PP and KDN9PP orthologs share a stringently conserved Arg residue that forms a salt bridge with the substrate carboxylate group. The split of the KDN9PP lineage from the KDO8PP orthologs is easily tracked by the acquisition of a Glu/Lys pair that supports KDN9P binding. Moreover, independently evolved lineages of KDO8PP orthologs exist, and are separated by diffuse active-site sequence boundaries. We infer a high tolerance of the KDO8PP catalytic platform to amino acid replacements that in turn influence substrate specificity changes and thereby facilitate the divergence in biological function.

The Crystal Structure of Arabidopsis VSP1 Reveals the Plant Class C-Like Phosphatase Structure of the DDDD Superfamily of Phosphohydrolases

Arabidopsis thaliana vegetative storage proteins, VSP1 and VSP2, are acid phosphatases and belong to the haloacid dehalogenase (HAD) superfamily. In addition to their potential nutrient storage function, they were thought to be involved in plant defense and flower development. To gain insights into the architecture of the protein and obtain clues about its function, we have tested their substrate specificity and solved the structure of VSP1. The acid phosphatase activities of these two enzymes require divalent metal such as magnesium ion. Conversely, the activity of these two enzymes is inhibited by vanadate and molybdate, but is resistant to inorganic phosphate. Both VSP1 and VSP2 did not exhibit remarkable activities to any physiological substrates tested. In the current study, we presented the crystal structure of recombinant VSP1 at 1.8 Å resolution via the selenomethionine single-wavelength anomalous diffraction (SAD). Specifically, an α-helical cap domain on the top of the α/β core domain is found to be involved in dimerization. In addition, despite of the low sequence similarity between VSP1 and other HAD enzymes, the core domain of VSP1 containing conserved active site and catalytic machinery displays a classic haloacid dehalogenase fold. Furthermore, we found that VSP1 is distinguished from bacterial class C acid phosphatase P4 by several structural features. To our knowledge, this is the first study to reveal the crystal structure of plant vegetative storage proteins.

Biosynthetic pathway toward carbohydrate-like moieties of alnumycins contains unusual steps for C-C bond formation and cleavage

Carbohydrate moieties are important components of natural products, which are often imperative for the solubility and biological activity of the compounds. The aromatic polyketide alnumycin A contains an extraordinary sugar-like 4'-hydroxy-5'-hydroxymethyl-2',7'-dioxane moiety attached via a carbon-carbon bond to the aglycone. Here we have extensively investigated the biosynthesis of the dioxane unit through (13)C labeling studies, gene inactivation experiments and enzymatic synthesis. We show that AlnA and AlnB, members of the pseudouridine glycosidase and haloacid dehalogenase enzyme families, respectively, catalyze C-ribosylation conceivably through Michael-type addition of d-ribose-5-phosphate and dephosphorylation. The ribose moiety may be attached both in furanose (alnumycin C) and pyranose (alnumycin D) forms. The C(1')-C(2') bond of alnumycin C is subsequently cleaved and the ribose unit is rearranged into an unprecedented dioxolane (cis-bicyclo[3.3.0]-2',4',6'-trioxaoctan-3'β-ol) structure present in alnumycin B. The reaction is catalyzed by Aln6, which belongs to a previously uncharacterized enzyme family. The conversion was accompanied with consumption of O(2) and formation of H(2)O(2), which allowed us to propose that the reaction may proceed via hydroxylation of C1' followed by retro-aldol cleavage and acetal formation. Interestingly, no cofactors could be detected and the reaction was also conducted in the presence of metal chelating agents. The last step is the conversion of alnumycin B into the final end-product alnumycin A catalyzed by Aln4, an NADPH-dependent aldo-keto reductase. This characterization of the dioxane biosynthetic pathway sets the basis for the utilization of C-C bound ribose, dioxolane and dioxane moieties in the generation of improved biologically active compounds.

Divergence of structure and function in the haloacid dehalogenase enzyme superfamily: Bacteroides thetaiotaomicron BT2127 is an inorganic pyrophosphatase.

The explosion of protein sequence information requires that current strategies for function assignment evolve to complement experimental approaches with computationally based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown haloalkanoate dehalogenase superfamily member BT2127 (Uniprot accession code Q8A5 V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics-structure-mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with a k(cat)/K(m) value for pyrophosphate of ~1 × 10(5) M(-1) s(-1)), together with the gene context, support the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure-guided site-directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. On the basis of this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172, and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue, β-phosphoglucomutase, control the leaving group size (phosphate vs glucose phosphate) and the position of the Asp acid/base in the open versus closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.

Analysis of the Structural Determinants Underlying Discrimination between Substrate and Solvent in β-Phosphoglucomutase Catalysis

Tauhe beta-phosphoglucomutase (beta-PGM) of the haloacid dehalogenase enzyme superfamily (HADSF) catalyzes the conversion of beta-glucose 1-phosphate (betaG1P) to glucose 6-phosphate (G6P) using Asp8 of the core domain active site to mediate phosphoryl transfer from beta-glucose 1,6-(bis)phosphate (betaG1,6bisP) to betaG1P. Herein, we explore the mechanism by which hydrolysis of the beta-PGM phospho-Asp8 is avoided during the time that the active site must remain open to solvent to allow the exchange of the bound product G6P with the substrate betaG1P. On the basis of structural information, a model of catalysis is proposed in which the general acid/base (Asp10) side chain moves from a position where it forms a hydrogen bond to the Thr16-Ala17 portion of the domain-domain linker to a functional position where it forms a hydrogen bond to the substrate leaving group O and a His20-Lys76 pair of the cap domain. This repositioning of the general acid/base within the core domain active site is coordinated with substrate-induced closure of the cap domain over the core domain. The model predicts that Asp10 is required for general acid/base catalysis and for stabilization of the enzyme in the cap-closed conformation. It also predicts that hinge residue Thr16 plays a key role in productive domain-domain association, that hydrogen bond interaction with the Thr16 backbone amide NH group is required to prevent phospho-Asp8 hydrolysis in the cap-open conformation, and that the His20-Lys76 pair plays an important role in substrate-induced cap closure. The model is examined via kinetic analyses of Asp10, Thr16, His20, and Lys76 site-directed mutants. Replacement of Asp10 with Ala, Ser, Cys, Asn, or Glu resulted in no observable activity. The kinetic consequences of the replacement of linker residue Thr16 with Pro include a reduced rate of Asp8 phosphorylation by betaG1,6bisP, a reduced rate of cycling of the phosphorylated enzyme to convert betaG1P to G6P, and an enhanced rate of phosphoryl transfer from phospho-Asp8 to water. The X-ray crystal structure of the T16P mutant at 2.7 A resolution provides a snapshot of the enzyme in an unnatural cap-open conformation where the Asp10 side chain is located in the core domain active site. The His20 and Lys76 site-directed mutants exhibit reduced activity in catalysis of the Asp8-mediated phosphoryl transfer between betaG1,6bisP and betaG1P but no reduction in the rate of phospho-Asp8 hydrolysis. Taken together, the results support a substrate induced-fit model of catalysis in which betaG1P binding to the core domain facilitates recruitment of the general acid/base Asp10 to the catalytic site and induces cap closure.

The X‐ray crystallographic structure and activity analysis of a Pseudomonas‐specific subfamily of the HAD enzyme superfamily evidences a novel biochemical function

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO_2114, from the plant pathogen Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO_2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO_2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO_2114 was confirmed by kinetic assays. To explore PSPTO_2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO_2114 homologs were mapped onto the PSPTO_2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst.

Structural Snapshots of Escherichia coli Histidinol Phosphate Phosphatase along the Reaction Pathway

HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of l-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn(2+) ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg(2+), Mn(2+), Co(2+), or Zn(2+), but Ca(2+) has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg(2+) ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca(2+).

Diversification of function in the haloacid dehalogenase enzyme superfamily: The role of the cap domain in hydrolytic phosphorus [sbnd]carbon bond cleavage

Phosphonatase functions in the 2-aminoethylphosphonate (AEP) degradation pathway of bacteria, catalyzing the hydrolysis of the C P bond in phosphonoacetaldehyde (Pald) via formation of a bi-covalent Lys53ethylenamine/Asp12 aspartylphosphate intermediate. Because phosphonatase is a member of the haloacid dehalogenase superfamily, a family predominantly comprised of phosphatases, the question arises as to how this new catalytic activity evolved. The source of general acid-base catalysis for Schiff-base formation and aspartylphosphate hydrolysis was probed using pH-rate profile analysis of active-site mutants and X-ray crystallographic analysis of modified forms of the enzyme. The 2.9 Å X-ray crystal structure of the mutant Lys53Arg complexed with Mg 2+ and phosphate shows that the equilibrium between the open and the closed conformation is disrupted, favoring the open conformation. Thus, proton dissociation from the cap domain Lys53 is required for cap domain–core domain closure. The likely recipient of the Lys53 proton is a water-His56 pair that serves to relay the proton to the carbonyl oxygen of the phosphonoacetaldehyde (Pald) substrate upon addition of the Lys53. The pH-rate profile analysis of active-site mutants was carried out to test this proposal. The proximal core domain residues Cys22 and Tyr128 were ruled out, and the role of cap domain His56 was supported by the results. The X-ray crystallographic structure of wild-type phosphonatase reduced with NaBH 4 in the presence of Pald was determined at 2.4 Å resolution to reveal Nε-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C(2) of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain Nε-ethylenamine-Lys53 functions as a general base in the hydrolysis of the aspartylphosphate bi-covalent enzyme intermediate. Because the enzyme residues proposed to play a key role in P C bond cleavage are localized on the cap domain, this domain appears to have evolved to support the diversification of the HAD phosphatase core domain for catalysis of hydrolytic P C bond cleavage.