Bichitra K. Biswal

The molecular structure of epoxide hydrolase B from Mycobacterium tuberculosis and its complex with a urea-based inhibitor.

Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC(50) approximately 19 nM) urea-based inhibitor at 2.1 and 2.4 A resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical alpha/beta hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the binding-site residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120 degrees about its C(alpha)-C(beta) bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the alpha/beta type.

Cloning, expression, purification, crystallization and preliminary X-ray studies of epoxide hydrolases A and B from Mycobacterium tuberculosis

Mycobacterium tuberculosis epoxide hydrolases A and B, corresponding to open reading frames Rv3617 and Rv1938, are detoxification enzymes against epoxides. The recombinant forms of these enzymes have been expressed in Escherichia coli and purified to homogeneity. Diffraction-quality crystals of Rv3617 and Rv1938 were obtained by the hanging-drop vapour-diffusion technique. Crystals of Rv3617 and Rv1938 diffracted to 3.0 and 2.1 A resolution, respectively, at the ALS synchrotron at Berkeley, CA, USA.

Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction studies of histidinol phosphate aminotransferase (HisC2) from Mycobacterium tuberculosis.

HisC2 from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion technique from a condition consisting of 7 mg ml(-1) HisC2 (in 20 mM Tris pH 8.8, 50 mM NaCl and 5% glycerol), 1 M succinic acid pH 7.0, 0.1 M HEPES pH 7.0 and 1%(w/v) polyethylene glycol monomethyl ether 2000. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 255.98, b=77.09, c = 117.97 Å. X-ray diffraction data were recorded to 2.45 Å resolution from a single crystal using the in-house X-ray facility.

HisB from Mycobacterium tuberculosis: cloning, overexpression in Mycobacterium smegmatis, purification, crystallization and preliminary X-ray crystallographic analysis

HisB, encoded by open reading frame Rv1601, possesses enzymatic activity as an imidazoleglycerol-phosphate dehydratase in the histidine-biosynthetic pathway of Mycobacterium tuberculosis. A recombinant form of HisB was crystallized in three crystal forms: crystals grown using 20% PEG 1500 as a precipitant belonged to either the cubic space group P432 or the tetragonal space group P4, while an orthorhombic crystal form belonging to space group P2(1)2(1)2 was obtained using 15% PEG 5000 and 10 mM MnCl(2) as precipitant. The structure of HisB in the orthorhombic crystal form was solved by the molecular-replacement method using the crystal structure of its Arabidopsis thaliana counterpart, which shares 47% sequence identity with Rv1601, as the search model.

Overexpression, purification, crystallization and structure determination of AspB, a putative aspartate aminotransferase from Mycobacterium tuberculosis

A recombinant version of a putative aspartate aminotransferase, AspB (encoded by the ORF Rv3565), from Mycobacterium tuberculosis (Mtb) was overexpressed in M. smegmatis and purified to homogeneity using liquid chromatography. Crystals of AspB were grown in a condition consisting of 0.2 M ammonium phosphate monobasic, 0.1 M calcium chloride dihydrate employing the hanging-drop vapour-diffusion method at 298 K. The crystals diffracted to a limit of 2.50 Å resolution and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=93.27, b=98.19, c=198.70 Å. The structure of AspB was solved by the molecular-replacement method using a putative aminotransferase from Silicibacter pomeroyi (PDB entry 3h14) as the search model. The template shares 46% amino-acid sequence identity with Mtb AspB. The crystal asymmetric unit contains four AspB molecules (the Mr of each is 42,035 Da).

Sample preparation, crystallization and structure solution of HisC from Mycobacterium tuberculosis

Histidinolphosphate aminotransferase (HisC; Rv1600) from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals suitable for X-ray analysis were grown by the hanging-drop vapour-diffusion technique using 30% polyethylene glycol monomethyl ether 2000 as the precipitant. The crystals belonged to the hexagonal space group P3221, with an unusual high solvent content of 74.5%. X-ray diffraction data were recorded to 3.08 Å resolution from a single crystal using in-house Cu Kα radiation. The structure of HisC was solved by the molecular-replacement method using its Corynebacterium glutamicum counterpart as a search model. HisC is a dimer in the crystal as well as in solution.

Histidinol phosphate phosphatase of Mtb: structural and functional analysis

The global tuberculosis (TB) epidemic, caused by the pathogen Mycobacterium tuberculosis (Mtb), is aggravated by the emergence of extremely drug resistant and multi-drug resistant strains. The present situation necessitates the identification and characterization of new drug targets. The histidine (His) biosynthetic pathway (Figure), which converts 5-phosphoribosyl-1pyrophosphate to histidine in ten enzymatic steps, is essential for Mtb growth but is absent in mammals, thus making it an attractive anti-TB drug target. The enzymes of the His pathway are largely conserved across the bacteria, fungi and lower eukaryotes that synthesize histidine de novo with some differences in a few catalytic steps; particularly the fifth, sixth and eighth steps. The determination of the Mtb genome sequence provided a map of the genes involved in its His pathway. However, it was unclear which particular enzyme catalyzes the eighth stepthe dephosphorylation of Histidinol phosphate(HOLP) to Histidinol(HOL). In this study, employing bioinformatic approaches, the Histidinol phosphate phosphatase of Mtb was identified. The target protein was overexpressed in M. smegmatis and purified to homogenity. Its function was established using biochemical assays. The protein was crystallized in native as well as in substrate-bound complex forms and their structures were determined. Residues that line the active site pocket and the residues that are involved in catalysis were determined using both crystal structure and mutational studies data. The biological functional unit of HisN is a dimer, the overall structure of a monomer (260 amino acids) folds into two distinct structural domains, N and C-terminal domains. A loop of 20-residue long connects these two domains. The N-terminal domain consists of residues 2-130. Its tertiary structure comprised of two long alpha helices followed by a six-stranded anti-parallel β-sheet. The C-terminal domain comprised of residues 150-260 and folds into a globular structure consisting a five-stranded anti-parallel β-sheet sandwiched between six α-helices.

Cloning, overexpression, purification, crystallization, and preliminary X-ray studies of SP_0149, the substrate binding protein of an ABC transporter from Streptococcus pneumoniae

A truncated (29 residues from the N-terminus) and N-terminal His-tagged form of SP_0149 from pneumococcal strain ATCC BAA-334 was overexpressed and purified to homogeneity using affinity and gel-filtration chromatography. Diffraction quality crystals were grown at 293 K using the hanging-drop vapour-diffusion technique. X-ray diffraction data were collected to 2.3 Å resolution from a single-crystal that belonged to the orthorhombic space group P2(1)2(1)2(1) with the unit-cell parameters a=54.56, b=75.61, c=75.52 Å. The calculated values of the Matthews coefficient assuming one molecule (with calculated molecular weight of 30 400 Da) in the crystal asymmetric unit and the corresponding solvent content were 2.56 Å3 Da(-1) and 52.0%, respectively.