Structure and mechanism of bacterial dehalogenases: different ways to cleave a carbon-halogen bond

The dehalogenases make use of fundamentally different strategies to cleave carbon-halogen bonds. The structurally characterized haloalkane dehalogenases, haloacid dehalogenases and 4-chlorobenzoate-coenzyme A dehalogenases use substitution mechanisms that proceed via a covalent aspartyl intermediate. Recent X-ray crystallographic analysis of a haloalcohol dehalogenase and a trans-3-chloroacrylic acid dehalogenase has provided detailed insight into a different intramolecular substitution mechanism and a hydratase-like mechanism, respectively. The available information on the various dehalogenases supports different views on the possible evolutionary origins of their activities.     

Crystal structures of the substrate free-enzyme, and reaction intermediate of the HAD superfamily member, haloacid dehalogenase DehIVa from Burkholderia cepacia MBA4

DehIVa is a haloacid dehalogenase (EC 3.8.1.2) from the soil and water borne bacterium Burkholderia cepacia MBA4, which belongs to the functionally variable haloacid dehalogenase (HAD) superfamily of enzymes. The haloacid dehalogenases catalyse the removal of halides from haloacids resulting in a hydroxlated product. These enzymes are of interest for their potential to degrade recalcitrant halogenated environmental pollutants and their use in the synthesis of industrial chemicals. The haloacid dehalogenases utilise a nucleophilic attack on the substrate by an aspartic acid residue to form an enzyme-substrate ester bond and concomitantly cleaving of the carbon-halide bond and release of a hydroxylated product following ester hydrolysis. We present the crystal structures of both the substrate-free DehIVa refined to 1.93 A resolution and DehIVa covalently bound to l-2-monochloropropanoate trapped as a reaction intermediate, refined to 2.7 A resolution. Electron density consistent with a previously unidentified yet anticipated water molecule in the active site poised to donate its hydroxyl group to the product and its proton to the catalytic Asp11 is evident. It has been unclear how substrate enters the active site of this and related enzymes. The results of normal mode analysis (NMA) are presented and suggest a means whereby the predicted global dynamics of the enzyme allow for entry of the substrate into the active site. In the context of these results, the possible role of Arg42 and Asn178 in a "lock down" mechanism affecting active site access is discussed. In silico substrate docking of enantiomeric substrates has been examined in order to evaluate the enzymes enantioselectivity.     

Structure- and function-based characterization of a new phosphoglycolate phosphatase from Thermoplasma acidophilum

The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel beta-sheet with strand order S10, S9, S8, S1, S2 and a small beta-hairpin, strands S3 and S4. This central sheet is flanked by a set of three alpha-helices on one side and two helices on the other. The smaller domain is composed of an open faced beta-sandwich represented by three antiparallel beta-strands, S5, S6, and S7, flanked by two oppositely oriented alpha-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl- ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase.     

Sequence‐ and activity‐based screening of microbial genomes for novel dehalogenases

Dehalogenases are environmentally important enzymes that detoxify organohalogens by cleaving their carbon-halogen bonds. Many microbial genomes harbour enzyme families containing dehalogenases, but a sequence-based identification of genuine dehalogenases with high confidence is challenging because of the low sequence conservation among these enzymes. Furthermore, these protein families harbour a rich diversity of other enzymes including esterases and phosphatases. Reliable sequence determinants are necessary to harness genome sequencing-efforts for accelerating the discovery of novel dehalogenases with improved or modified activities. In an attempt to extract dehalogenase sequence fingerprints, 103 uncharacterized potential dehalogenase candidates belonging to the α/β hydrolase (ABH) and haloacid dehalogenase-like hydrolase (HAD) superfamilies were screened for dehalogenase, esterase and phosphatase activity. In this first biochemical screen, 1 haloalkane dehalogenase, 1 fluoroacetate dehalogenase and 5 l -2-haloacid dehalogenases were found (success rate 7%), as well as 19 esterases and 31 phosphatases. Using this functional data, we refined the sequence-based dehalogenase selection criteria and applied them to a second functional screen, which identified novel dehalogenase activity in 13 out of only 24 proteins (54%), increasing the success rate eightfold. Four new l -2-haloacid dehalogenases from the HAD superfamily were found to hydrolyse fluoroacetate, an activity never previously ascribed to enzymes in this superfamily.     

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.     

Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima

We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 A resolution by selenomethionine single-wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose-6-phosphate phosphatase and sucrose-6(F)-phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 x 40 x 35 A(3), and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six-stranded beta-sheet surrounded by four alpha-helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.     

Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene.

The haloacid dehalogenase of the 1,2-dichloroethane-utilizing bacterium Xanthobacter autotrophicus GJ10 was purified from a mutant with an eightfold increase in expression of the enzyme. The mutant was obtained by selecting for enhanced resistance to monobromoacetate. The enzyme was purified through (NH4)2SO4 fractionation, DEAE-cellulose chromatography, and hydroxylapatite chromatography. The molecular mass of the protein was 28 kDa as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 36 kDa as determined with gel filtration on Superose 12 fast protein liquid chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhlB) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10.     

Engineering a catabolic pathway in plants for the degradation of 1,2-dichloroethane

Plants are increasingly being employed to clean up environmental pollutants such as heavy metals; however, a major limitation of phytoremediation is the inability of plants to mineralize most organic pollutants. A key component of organic pollutants is halogenated aliphatic compounds that include 1,2-dichloroethane (1,2-DCA). Although plants lack the enzymatic activity required to metabolize this compound, two bacterial enzymes, haloalkane dehalogenase (DhlA) and haloacid dehalogenase (DhlB) from the bacterium Xanthobacter autotrophicus GJ10, have the ability to dehalogenate a range of halogenated aliphatics, including 1,2-DCA. We have engineered the dhlA and dhlB genes into tobacco (Nicotiana tabacum 'Xanthi') plants and used 1,2-DCA as a model substrate to demonstrate the ability of the transgenic tobacco to remediate a range of halogenated, aliphatic hydrocarbons. DhlA converts 1,2-DCA to 2-chloroethanol, which is then metabolized to the phytotoxic 2-chloroacetaldehyde, then chloroacetic acid, by endogenous plant alcohol dehydrogenase and aldehyde dehydrogenase activities, respectively. Chloroacetic acid is dehalogenated by DhlB to produce the glyoxylate cycle intermediate glycolate. Plants expressing only DhlA produced phytotoxic levels of chlorinated intermediates and died, while plants expressing DhlA together with DhlB thrived at levels of 1,2-DCA that were toxic to DhlA-expressing plants. This represents a significant advance in the development of a low-cost phytoremediation approach toward the clean-up of halogenated organic pollutants from contaminated soil and groundwater.     

Haloalkane dehalogenases: structure of a Rhodococcus enzyme

The hydrolytic haloalkane dehalogenases are promising bioremediation and biocatalytic agents. Two general classes of dehalogenases have been reported from Xanthobacter and Rhodococcus. While these enzymes share 30% amino acid sequence identity, they have significantly different substrate specificities and halide-binding properties. We report the 1.5 A resolution crystal structure of the Rhodococcus dehalogenase at pH 5.5, pH 7.0, and pH 5.5 in the presence of NaI. The Rhodococcus and Xanthobacter enzymes have significant structural homology in the alpha/beta hydrolase core, but differ considerably in the cap domain. Consistent with its broad specificity for primary, secondary, and cyclic haloalkanes, the Rhodococcus enzyme has a substantially larger active site cavity. Significantly, the Rhodococcus dehalogenase has a different catalytic triad topology than the Xanthobacter enzyme. In the Xanthobacter dehalogenase, the third carboxylate functionality in the triad is provided by D260, which is positioned on the loop between beta7 and the penultimate helix. The carboxylate functionality in the Rhodococcus catalytic triad is donated from E141. A model of the enzyme cocrystallized with sodium iodide shows two iodide binding sites; one that defines the normal substrate and product-binding site and a second within the active site region. In the substrate and product complexes, the halogen binds to the Xanthobacter enzyme via hydrogen bonds with the N(eta)H of both W125 and W175. The Rhodococcusenzyme does not have a tryptophan analogous to W175. Instead, bound halide is stabilized with hydrogen bonds to the N(eta)H of W118 and to N(delta)H of N52. It appears that when cocrystallized with NaI the Rhodococcus enzyme has a rare stable S-I covalent bond to S(gamma) of C187.     

X-ray crystal structure of Mycobacterium tuberculosis haloalkane dehalogenase Rv2579

Haloalkane dehalogenases are enzymes well known to be important in bioremediation; the organisms from which they are produced are able to clean up toxic organohalides from polluted environments. However, besides being found in such contaminated environments, these enzymes have also been found in root or tissue-colonizing bacterial species. The haloalkane dehalogenase Rv2579 from Mycobacterium tuberculosis H37Rv has been cloned, expressed, purified and its crystal structure determined at high resolution (1.2A). In addition, the crystal structure of the enzyme has been determined in complex with the product from the reaction with 1,3-dibromopropane, i.e. 1,3-propanediol and in complex with the classical substrate of haloalkane dehalogenases, 1,2-dichloroethane. The enzyme is a two-domain protein having a catalytic domain of an alpha/beta hydrolase fold and a cap domain. The active site residues and the halide-stabilizing residues have been identified as Asp109, Glu133, His273, Asn39 and Trp110. Its overall structure is similar to those of other known haloalkane dehalogenases. Its mechanism of action involves an SN2 nucleophilic displacement.     

Structure prediction and evolution of a halo-acid dehalogenase of Burkholderia mallei

Environmental pollutants containing halogenated organic compounds e.g. haloacid, can cause a plethora of health problems. The structural and functional analyses of the gene responsible of their degradation are an important aspect for environmental studies and are important to human well-being. It has been shown that some haloacids are toxic and mutagenic. Microorganisms capable of degrading these haloacids can be found in the natural environment. One of these, a soil-borne Burkholderia mallei posses the ability to grow on monobromoacetate (MBA). This bacterium produces a haloacid dehalogenase that allows the cell to grow on MBA, a highly toxic and mutagenic environmental pollutant. For the structural and functional analysis, a 346 amino acid encoding protein sequence of haloacid dehalogenase is retrieve from NCBI data base. Primary and secondary structure analysis suggested that the high percentage of helices in the structure makes the protein more flexible for folding, which might increase protein interactions. The consensus protein sub-cellular localization predictions suggest that dehalogenase protein is a periplasmic protein 3D2GO server, suggesting that it is mainly employed in metabolic process followed by hydrolase activity and catalytic activity. The tertiary structure of protein was predicted by homology modeling. The result suggests that the protein is an unstable protein which is also an important characteristic of active enzyme enabling them to bind various cofactors and substrate for proper functioning. Validation of 3D structure was done using Ramachandran plot ProsA-web and RMSD score. This predicted information will help in better understanding of mechanism underlying haloacid dehalogenase encoding protein and its evolutionary relationship.     

The Structural Domains of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Cooperate in Substrate Hydrolysis: 3D Structure and Enzymatic Mechanism

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It colonizes different tissues by the utilization of diverse mechanisms. One of these may involve the breakdown of the host cell membrane through the sequential action of hemolytic phospholipase C and phosphorylcholine phosphatase (PchP). The action of hemolytic phospholipase C on phosphatidylcholine produces phosphorylcholine, which is hydrolyzed to choline (Cho) and inorganic phosphate by PchP. The available biochemical data on this enzyme demonstrate the involvement of two Cho-binding sites in the catalytic cycle and in enzyme regulation. The crystal structure of P. aeruginosa PchP has been determined. It folds into three structural domains. The first domain harbors all the residues involved in catalysis and is well conserved among the haloacid dehalogenase superfamily of proteins. The second domain is characteristic of PchP and is involved in the recognition of the Cho moiety of the substrate. The third domain stabilizes the relative position of the other two. Fortuitously, the crystal structure of PchP captures molecules of Bistris (2‐[bis(2‐hydroxyethyl)amino]‐2‐(hydroxymethyl)propane‐1,3‐diol) at the active site and at an additional site. This represents two catalytically relevant complexes with just one or two inhibitory Bistris molecules and provides the basis of the PchP function and regulation. Site‐directed mutagenesis along with biochemical experiments corroborates the structural observations and demonstrates the interplay between different sites for Cho recognition and inhibition. The structural comparison of PchP with other phosphatases of the haloacid dehalogenase family provides a three‐dimensional picture of the conserved catalytic cycle and the structural basis for the recognition of the diverse substrate molecules.     

Biochemical and structural studies of a l-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii

Haloacid dehalogenases have potential applications in the pharmaceutical and fine chemical industry as well as in the remediation of contaminated land. The l-2-haloacid dehalogenase from the thermophilic archaeon Sulfolobus tokodaii has been cloned and over-expressed in Escherichia coli and successfully purified to homogeneity. Here we report the structure of the recombinant dehalogenase solved by molecular replacement in two different crystal forms. The enzyme is a homodimer with each monomer being composed of a core-domain of a β-sheet bundle surrounded by α-helices and an α-helical sub-domain. This fold is similar to previously solved mesophilic l-haloacid dehalogenase structures. The monoclinic crystal form contains a putative inhibitor l-lactate in the active site. The enzyme displays haloacid dehalogenase activity towards carboxylic acids with the halide attached at the C2 position with the highest activity towards chloropropionic acid. The enzyme is thermostable with maximum activity at 60°C and a half-life of over 1 h at 70°C. The enzyme is relatively stable to solvents with 25% activity lost when incubated for 1 h in 20% v/v DMSO.     

Genetics and biochemistry of 1,2-dichloroethane degradation

Dichloroethane (1,2-DCE) is a synthetic compound that is not known to be formed naturally. Nevertheless, several pure microbial cultures are able to use it as a sole carbon source for growth. Degradation of 1,2-DCE proceeds via 2-chloroethanol, chloroacetaldehyde and chloroacetate to glycolate. The genes encoding the enzymes responsible for the conversion of 1,2-DCE to glycolic acid have been isolated. The haloalkane dehalogenase and an aldehyde dehydrogenase are plasmid encoded. Two other enzymes, the alcohol dehydrogenase and the haloacid dehalogenase, are chromosomally encoded. Sequence analysis indicates that the haloacid dehalogenase belongs to the L-specific 2-chloroproprionic acid dehalogenases. From the three-dimensional structure and sequence similarities, the haloalkane dehalogenase appears to be a member of the / hydrolase fold hydrolytic enzymes, of which several are involved in the degradation of aromatic and aliphatic xenobiotic compounds.     

Cloning and characterization of a cryptic haloacid dehalogenase from Burkholderia cepacia MBA4.

Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards the L-isomer of 2-monochloropropionic acid only. The structural gene, chd1, was isolated on a 1.7-kb PstI fragment. This fragment contains a functional promoter, because expression of chd1 in E. coli is orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence of chd1 did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.     

Diversification of function in the haloacid dehalogenase enzyme superfamily: The role of the cap domain in hydrolytic phosphoruscarbon 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 A X-ray crystal structure of the mutant Lys53Arg complexed with Mg2+ 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 NaBH4 in the presence of Pald was determined at 2.4A resolution to reveal N epsilon-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C2 of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain N epsilon-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.     

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.     

Structure of recombinant Haemophilus influenzae e (P4) acid phosphatase reveals a new member of the haloacid dehalogenase superfamily

Lipoprotein e (P4) from Haemophilus influenzae belongs to the "DDDD" superfamily of phosphohydrolases and is the prototype of class C nonspecific acid phosphatases. P4 is also a component of a H. influenzae vaccine. We report the crystal structures of recombinant P4 in the ligand-free and tungstate-inhibited forms, which are the first structures of a class C phosphatase. P4 has a two-domain architecture consisting of a core alpha/beta domain and a smaller alpha domain. The core domain features a five-stranded beta-sheet flanked by helices on both sides that is reminiscent of the haloacid dehalogenase superfamily. The alpha domain appears to be unique and plays roles in substrate binding and dimerization. The active site is solvent accessible and located in a cleft between the two domains. The structure shows that P4 is a metalloenzyme and that magnesium is the most likely metal ion in the crystalline recombinant enzyme. The ligands of the metal ion are the carboxyl groups of the first and third Asp residues of the DDDD motif, the backbone carbonyl of the second Asp of the DDDD motif, and two water molecules. The structure of the tungstate-bound enzyme suggests that Asp64 is the nucleophile that attacks the substrate P atom. Dimerization appears to be important for catalysis because intersubunit contacts stabilize the active site. Analysis of the structural context of mutations engineered for vaccine studies shows that the most promising mutations are located in the dimer interface. This observation suggests a structure-based vaccine design strategy in which the dimer interface is disrupted in order to expose epitopes that are buried in dimeric P4.     

Crystal structure of trehalose-6-phosphate phosphatase-related protein: biochemical and biological implications

We report here the crystal structure of a trehalose-6-phosphate phosphatase-related protein (T6PP) from Thermoplasma acidophilum, TA1209, determined by the dual-wavelength anomalous diffraction (DAD) method. T6PP is a member of the haloacid dehalogenase (HAD) superfamily with significant sequence homology with trehalose-6-phosphate phosphatase, phosphoserine phosphatase, P-type ATPases and other members of the family. T6PP possesses a core domain of known alpha/beta-hydrolase fold, characteristic of the HAD family, and a cap domain, with a tertiary fold consisting of a four-stranded beta-sheet with two alpha-helices on one side of the sheet. An active-site magnesium ion and a glycerol molecule bound at the interface between the two domains provide insight into the mode of substrate binding by T6PP. A trehalose-6-phosphate molecule modeled into a cage formed by the two domains makes favorable interactions with the protein molecule. We have confirmed that T6PP is a trehalose phosphatase from amino acid sequence, three-dimensional structure, and biochemical assays.     

Sec-dependent and Sec-independent translocation of haloacid dehalogenase Chd1 of Burkholderia cepacia MBA4 in Escherichia coli

2-Haloacid dehalogenases are hydrolytic enzymes that cleave the halogen-carbon bond(s) in haloalkanoic acids. We have previously isolated a cryptic haloacid dehalogenase gene from Burkholderia cepacia MBA4 and expressed it in Escherichia coli. This recombinant protein is unusual in having a long leader sequence, a property of periplasmic enzymes. In this paper, we report the functional role of this leader sequence. Western blot analyses showed that Chd1 is translocated to the periplasm. The results on the expression of Chd1 in the presence of sodium azide suggested the cleavage of the leader to be Sec-dependent. Chimeras of Chd1 and green fluorescent protein demonstrated that the leader sequence is fully functional in translocating the fusion protein to the periplasm. The expression of the chimeras in Sec mutants supported the Sec-dependent translocation. Surprisingly, recombinant Chd1 and a chimera with no leader sequence were also found in the periplasm.