Insights into the Regulatory Characteristics of the Mycobacterial Dephosphocoenzyme A Kinase: Implications for the Universal CoA Biosynthesis Pathway

Being vastly different from the human counterpart, we suggest that the last enzyme of the Mycobacterium tuberculosis Coenzyme A biosynthetic pathway, dephosphocoenzyme A kinase (CoaE) could be a good anti-tubercular target. Here we describe detailed investigations into the regulatory features of the enzyme, affected via two mechanisms. Enzymatic activity is regulated by CTP which strongly binds the enzyme at a site overlapping that of the leading substrate, dephosphocoenzyme A (DCoA), thereby obscuring the binding site and limiting catalysis. The organism has evolved a second layer of regulation by employing a dynamic equilibrium between the trimeric and monomeric forms of CoaE as a means of regulating the effective concentration of active enzyme. We show that the monomer is the active form of the enzyme and the interplay between the regulator, CTP and the substrate, DCoA, affects enzymatic activity. Detailed kinetic data have been corroborated by size exclusion chromatography, dynamic light scattering, glutaraldehyde crosslinking, limited proteolysis and fluorescence investigations on the enzyme all of which corroborate the effects of the ligands on the enzyme oligomeric status and activity. Cysteine mutagenesis and the effects of reducing agents on mycobacterial CoaE oligomerization further validate that the latter is not cysteine-mediated or reduction-sensitive. These studies thus shed light on the novel regulatory features employed to regulate metabolite flow through the last step of a critical biosynthetic pathway by keeping the latter catalytically dormant till the need arises, the transition to the active form affected by a delicate crosstalk between an essential cellular metabolite (CTP) and the precursor to the pathway end-product (DCoA).

Usefulness of in Situ Single Crystal to Single Crystal Transformation (SCSC) Studies in Understanding the Temperature-Dependent Dimensionality Cross-over and Structural Reorganization in Copper-Containing Metal-Organic Frameworks (MOFs)

Two copper-containing compounds [Cu(3)(mu(3)-OH)(2)-(H(2)O)(2){(SO(3))-C(6)H(3)-(COO)(2)}(CH(3)COO)] , I, and [Cu(5)(mu(3)-OH)(2)(H(2)O)(6){(NO(2))-C(6)H(3)-(COO)(2)}(4)]center dot 5H(2)O, II, were prepared using sulphoisophthalic and nitroisophthalic acids. The removal of the coordinated water molecules in the compounds was investigated using in situ single crystal to single crystal (SCSC) transformation studies, temperature-dependent powder X-ray diffraction (PXRD), and thermogravimetric analysis (TGA). The efficacy of SCSC transformation studies were established by the observation of dimensionality cross-over from a two-dimensional (I) to a three-dimensional structure, Cu(6)(mu(3)-OH)(4){(SO(3))-C(6)H(3)-(COO)(2)}(2)(CH(3)COO)(2), Ia, during the removal of the coordinated water molecules. Compound H exhibited a structural reorganization forming Cu(5)(mu(2)-OH)(2){(NO(2))C(6)H(3)-(COO)(2))(4)], Ha, possessing trimeric (Cu(3)O(12)) and dimeric (Cu(2)O(8)) copper clusters. The PXRD studies indicate that the three-dimensional structure (Ia) is transient and unstable, reverting back to the more stable two-dimensional structure (I) on cooling to room temperature. Compound Ha appears to be more stable at room temperature. The rehydration/dehydration studies using a modified TGA setup suggest complete rehydration of the water molecules, indicating that the water molecules in both compounds are labile. A possible model for the observed changes in the structures has been proposed. Magnetic studies indicate changes in the exchanges between the copper centers in Ha, whereas no such behavior was observed in Ia.

Computational tools for mechanistic discrimination in the reductive and metathesis coupling reactions mediated by titanium(IV) isopropoxide

A theoretical study has been carried out at the B3LYP/LANL2DZ level to compare the reactivity of phenyl isocyanate and phenyl isothiocyanate towards titanium(IV) alkoxides. Isocyanates are shown to favour both mono insertion and double insertion reactions. Double insertion in a head-to-tail fashion is shown to be more exothermic than double insertion in a head-to-head fashion. The head-to-head double insertion leads to the metathesis product, a carbodiimide, after the extrusion of carbon dioxide. In the case of phenyl isothiocyanate, calculations favour the formation of only mono insertion products. Formation of a double insertion product is highly unfavourable. Further, these studies indicate that the reverse reaction involving the metathesis of N,N-'-diphenyl carbodiimide with carbon dioxide is likely to proceed more efficiently than the metathesis reaction with carbon disulphide. This is in excellent agreement with experimental results as metathesis with carbon disulphide fails to occur. In a second study, multilayer MM/QM calculations are carried out on intermediates generated from reduction of titanium(IV) alkoxides to investigate the effect of alkoxy bridging on the reactivity of multinuclear Ti species. Bimolecular coupling of imines initiated by Ti(III) species leads to a mixture of diastereomers and not diastereoselective coupling of the imine. However if the reaction is carried out by a trimeric biradical species, diastereoselective coupling of the imine is predicted. The presence of alkoxy bridges greatly favours the formation of the d,l (+/-) isomer, whereas the intermediate without alkoxy bridges favours the more stable meso isomer. As a bridged trimeric species, stabilized by bridging alkoxy groups, correctly explains the diastereoselective reaction, it is the most likely intermediate in the reaction.

A Histidine Aspartate Ionic Lock Gates the Iron Passage in Miniferritins from Mycobacterium smegmatis

Background: DNA-binding protein from starved cells (Dps) are nano-compartments that can oxidize and store iron rendering protection from free radicals. Results: A histidine-aspartate ionic cluster in mycobaterial Dps2 modulates the rate of iron entry and exit in these proteins. Conclusion: Substitutions that disrupt the cluster interface alter the iron uptake/release properties with localized structural changes. Significance: Identifying important gating residues can help in designing nano-delivery vehicles. Dps (DNA-binding protein from starved cells) are dodecameric assemblies belonging to the ferritin family that can bind DNA, carry out ferroxidation, and store iron in their shells. The ferritin-like trimeric pore harbors the channel for the entry and exit of iron. By representing the structure of Dps as a network we have identified a charge-driven interface formed by a histidine aspartate cluster at the pore interface unique to Mycobacterium smegmatis Dps protein, MsDps2. Site-directed mutagenesis was employed to generate mutants to disrupt the charged interactions. Kinetics of iron uptake/release of the wild type and mutants were compared. Crystal structures were solved at a resolution of 1.8-2.2 for the various mutants to compare structural alterations vis a vis the wild type protein. The substitutions at the pore interface resulted in alterations in the side chain conformations leading to an overall weakening of the interface network, especially in cases of substitutions that alter the charge at the pore interface. Contrary to earlier findings where conserved aspartate residues were found crucial for iron release, we propose here that in the case of MsDps2, it is the interplay of negative-positive potentials at the pore that enables proper functioning of the protein. In similar studies in ferritins, negative and positive patches near the iron exit pore were found to be important in iron uptake/release kinetics. The unique ionic cluster in MsDps2 makes it a suitable candidate to act as nano-delivery vehicle, as these gated pores can be manipulated to exhibit conformations allowing for slow or fast rates of iron release.

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.

Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers that drives fusion of viral and cellular membranes. While it is expected that native Env is a good immunogen, its metastability results in exposure of immunodominant nonneutralizing epitopes. In the present study, we stabilize native Env trimers by incorporation of a number of different mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The stabilized constructs described here can be incorporated into DNA vaccine candidates.

Stabilization of Co-3 - Oxoclusters in a pcu Net: Synthesis, Structure, Solvent Exchange (Single Crystal to Single Crystal) and Magnetic Studies

The solvothermal reaction of CoCl(2)4H(2)O and 4,4-sulfonyldibenzoic acid (H(2)SDBA) resulted in the formation of a three-dimensional coordination polymer Co-3(C14H8O6S)(3)(DMA)(2)(MeOH)].DMA (Ia) consisting of trinuclear Co-3 oxo-cluster units. The Co-3 trimeric units are connected by SDBA(2-) anions leading to a three dimensional structure with a pcu topology. The terminal methanol molecules could be exchanged in a single crystal to single crystal (SCSC) fashion by other similar solvent molecules (ethanol, acetonitrile, water, ethyleneglycol). Magnetic studies on the parent compound, Ia, indicate antiferromagnetic interactions between the central metal atoms.

Developmentally regulated transcription factors in Drosophila melanogaster

During early stages of Drosophila development the heat shock response cannot be induced. It is reasoned that the adverse effects on cell cycle and cell growth brought about by Hsp70 induction must outweigh the beneficial aspects of Hsp70 induction in the early embryo. Although the Drosophila heat shock transcription factor (dHSF) is abundant in the early embryo, it does not enter the nucleus in response to heat shock. In older embryos and in cultured cells the factor is localized within the nucleus in an apparent trimeric structure that binds DNA with high affinity. The domain responsible for nuclear localization upon stress resides between residues 390 and 420 of the dHSF. Using that domain as bait in a yeast two-hybrid system we now report the identification and cloning of a nuclear transport protein Drosophila karyopherin-α3(dKap- α3). Biochemical methods demonstrate that the dKap-α3 protein binds specifically to the dHSF's nuclear localization sequence (NLS). Furthermore, the dKap-α3 protein does not associate with NLSs that contain point mutations which are not transported in vivo. Nuclear docking studies also demonstrate specific nuclear targeting of the NLS substrate by dKap-α3.Consistant with previous studies demonstrating that early Drosophila embryos are refractory to heat shock as a result of dHSF nuclear exclusion, we demonstrate that the early embryo is deficient in dKap-α3 protein through cycle 12. From cycle 13 onward the transport factor is present and the dHSF is localized within the nucleus thus allowing the embryo to respond to heat shock.

The pair-rule gene fushi tarazu (ftz) is a well-studied zygotic segmentation gene that is necessary for the development of the even-numbered parasegments in Drosophila melanogastor. During early embryogenesis, ftz is expressed in a characteristic pattern of seven stripes, one in each of the even-numbered parasegments. With a view to understand how ftz is transcriptionally regulated, cDNAs that encode transcription factors that bind to the zebra element of the ftz promoter have been cloned. Chapter Ill reports the cloning and characterization of the eDNA encoding zeb-1 (zebra element binding protein), a novel steroid receptor-like molecule that specifically binds to a key regulatory element of the ftz promoter. In transient transfection assays employing Drosophila tissue culture cells, it has been shown that zeb-1 as well as a truncated zeb-1 polypeptide (zeb480) that lacks the putative ligand binding domain function as sequencespecific trans-activators of the ftz gene.

The Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Chapter IV reports the eDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in-situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20-40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in fourteen stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. The data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

Mechanisms of substrate selection by the signal recognition particle

The signal recognition particle (SRP) targets membrane and secretory proteins to their correct cellular destination with remarkably high fidelity. Previous studies have shown that multiple checkpoints exist within this targeting pathway that allows ‘correct cargo’ to be quickly and efficiently targeted and for ‘incorrect cargo’ to be promptly rejected. In this work, we delved further into understanding the mechanisms of how substrates are selected or discarded by the SRP. First, we discovered the role of the SRP fingerloop and how it activates the SRP and SRP receptor (SR) GTPases to target and unload cargo in response to signal sequence binding. Second, we learned how an ‘avoidance signal’ found in the bacterial autotransporter, EspP, allows this protein to escape the SRP pathway by causing the SRP and SR to form a ‘distorted’ complex that is inefficient in delivering the cargo to the membrane. Lastly, we determined how Trigger Factor, a co-translational chaperone, helps SRP discriminate against ‘incorrect cargo’ at three distinct stages: SRP binding to RNC; targeting of RNC to the membrane via SRP-FtsY assembly; and stronger antagonism of SRP targeting of ribosomes bearing nascent polypeptides that exceed a critical length. Overall, results delineate the rich underlying mechanisms by which SRP recognizes its substrates, which in turn activates the targeting pathway and provides a conceptual foundation to understand how timely and accurate selection of substrates is achieved by this protein targeting machinery.

Structural Characterizations of the Dimeric Anti-HIV Antibody 2G12 and the HIV-2 Envelope Glycoprotein

More than thirty years after the discovery that Human Immunodeficiency Virus (HIV) was the causative agent of Acquired Immunodeficiency Syndrome (AIDS), the disease remains pandemic as long as no effective universal vaccine is found. Over 34 million individuals in the world are infected with the virus, and the vast majority of them have no access to the antiretroviral therapies that have largely reduced HIV to a chronic disease in the developed world. The first chapter of this thesis introduces the history of the virus. The key to the infectious mechanism of the virus lies in its envelope glycoprotein (Env), a trimeric spike on the viral surface that utilizes host T cell receptors for entry. Though HIV-1 Env is immunogenic, most infected patients do not mount an effective neutralizing antibody response against it. Broadly-neutralizing anti-Env antibodies (bNAbs) present in the serum of a minority of infected individuals are usually sufficient to prevent the progression to full blown AIDS. Thus, the molecular details of these bNAbs as well as the antibody-antigen interface are of prime interest for structural studies, as insight gained would contribute to the design of a more effective immunogen and potential vaccine candidate. The second chapter of this thesis describes the low-resolution crystal structure of one such antibody, 2G12 dimer, which targets a high mannose epitope on the surface of Env. Patients infected with HIV-2, a related virus with ~35% sequence identity in the Env region, can generally mount a robust antibody response sufficient for viral control for reasons still unknown. The final two chapters of this thesis focus on the first reported structural studies of HIV-2 Env, the molecular details of which may inform HIV-1 therapy and immunogen design.

New high-dimensional networks based on polyoxometalate and crown ether building blocks

Three novel supramolecular assemblies constructed from polyoxometalate and crown ether building blocks, [(DB18C6)Na(H2O)(1.5)](2)Mo6O19.CH3CN, 1, and [{Na(DB18C6)(H2O)(2)}(3)(H2O)(2)]XMo12O40.6DMF.CH3CN (X = P, 2, and As, 3; DB18C6 = dibenzo-18-crown-6; DMF = N,N-dimethylfomamide), have been synthesized and characterized by elemental analyses, IR, UV-vis, EPR, TG, and single crystal X-ray diffraction. Compound 1 crystallizes in the tetragonal space group P4/mbm with a = 16.9701(6) Angstrom, c = 14.2676(4) Angstrom, and Z = 2. Compound 2 crystallizes in the hexagonal space group P6(3)/m with a = 15,7435(17) Angstrom, c = 30.042(7) Angstrom, gamma = 120degrees, and Z = 2. Compound 3 crystallizes in the hexagonal space group P6(3)/m with a = 15.6882(5) Angstrom, c = 29.9778(18) Angstrom, gamma = 120degrees, and Z = 2. Compound 1 exhibits an unusual three-dimensional network with one-dimensional sandglasslike channels based on the extensive weak forces between the oxygen atoms on the [Mo6O19](2-) polyoxoanions and the CH2 groups of crown ether molecules, Compounds 2 and 3 are isostructural, and both contain a novel semiopen cagelike trimeric cation [{Na(DB18C6)(H2O)(2)}(3)(H2O)(2)](3+). In their packing arrangement, an interesting 2-D "honeycomblike" "host" network is formed, in which the [XMo12O40](3-) (X = As and P) polyoxoanion "guests" resided.

The structure of Aquifex aeolicus sulfide:quinone oxidoreductase, a basis to understand sulfide detoxification and respiration

Sulfide: quinone oxidoreductase (SQR) is a flavoprotein with homologues in all domains of life except plants. It plays a physiological role both in sulfide detoxification and in energy transduction. We isolated the protein from native membranes of the hyperthermophilic bacterium Aquifex aeolicus, and we determined its X-ray structure in the "as-purified,'' substrate-bound, and inhibitor-bound forms at resolutions of 2.3, 2.0, and 2.9 angstrom, respectively. The structure is composed of 2 Rossmann domains and 1 attachment domain, with an overall monomeric architecture typical of disulfide oxidoreductase flavoproteins. A. aeolicus SQR is a surprisingly trimeric, periplasmic integral monotopic membrane protein that inserts about 12 angstrom into the lipidic bilayer through an amphipathic helix-turn-helix tripodal motif. The quinone is located in a channel that extends from the si side of the FAD to the membrane. The quinone ring is sandwiched between the conserved amino acids Phe-385 and Ile-346, and it is possibly protonated upon reduction via Glu-318 and/or neighboring water molecules. Sulfide polymerization occurs on the re side of FAD, where the invariant Cys-156 and Cys-347 appear to be covalently bound to polysulfur fragments. The structure suggests that FAD is covalently linked to the polypeptide in an unusual way, via a disulfide bridge between the 8-methyl group and Cys-124. The applicability of this disulfide bridge for transferring electrons from sulfide to FAD, 2 mechanisms for sulfide polymerization and channeling of the substrate, S2-, and of the product, S-n, in and out of the active site are discussed.

Characterization of the main light-harvesting chlorophyll a/b-protein complex of green alga, Bryopsis corticulans

The main light-harvesting chlorophyll a/b -protein complex (LHC II) has been isolated directly from thylakoid membranes of shiphonous green alga, Bryopsis corticulans Setch. by using two consecutive runs of anion exchange and gel-filtration chromatography. Monomeric and trimeric subcomplexes of LHC 11 were obtained by using sucrose gradient ultracentrifugation. Pigment analysis by reversed-phase high performance liquid chromatography showed that chlorophyll a (Chl a), chlorophyll b (Chl b), neoxanthin, violaxanthin and siphonaxanthin were involved in LHC 11 from B. corticulans. The properties of electronic transition of monomeric LHC II showed similarities to those of trimeric LHC II. Circular dichroism spectroscopy showed that strong intramolecular interaction of excitonic dipoles between Chl a and between Chl b exist in one LHC II apoprotein, while the intermolecular interaction of these dipoles can be intensified in the trimeric structure. The monomer has high efficient energy transfer from Chl b and siphonaxanthin to Chl a similarly to that of the trimer. Our results suggest that in B. corticulans, LHC II monomer has high ordered pigment organization that play effective physiological function as the trimer, and thus it might be also a functional organization existing in thylakoid membrane of B. corticulans.

Isolation of the main light-harvesting chlorophyll a/b-protein complex from thylakoid membranes of marine alga, Bryopsis corticulans by a direct method

The main chlorophyll a/b light-harvesting complex (LHC 11) has been isolated directly from thylakoid membranes of marine green alga (Bryopsis corticulans Setch.) by two consecutive runs of anion exchange and gel-filtration chromatography. LHC 11 proteins in the membrane extracts treated with 3% n-Octyl-b-D-glucopyranoside (OG) obtained specific binding ability on Q Sepharose column, and thus were isolated from the thylakoid membranes in a highly selective fraction. The monomeric, trimeric and oligomeric subcomplexes of LHC 11 have been obtained by fractionation of the LHC 11 mixes with sucrose density gradient ultracentrifugation. The SDS-PAGE analysis of peptide composition and absorption spectrum showed that LHC 11 monomers, trimers and oligomers prepared through this work were intact and in high purity. Our report is the first to show that it is possible to purify LHC If directly from thylakoid membranes without extensively biochemical purification.

Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E(mM) for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of alpha-helix (56%) with lesser amounts of random coil (21%), beta-turn (13%) and beta-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% alpha-helix, 33.7% random coil and 10.5% beta-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60 degrees C for 5 min; thereafter by cooling at 1 degrees C min(-1) to 45 degrees C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60 degrees C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330-350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.