PDBsum entry 1w0d

Oxidoreductase

PDB id: 1w0d

Contents

Protein chains

337 a.a. *

Ligands

SO4

Waters ×579

* Residue conservation analysis

PDB id:

1w0d

Name:

Oxidoreductase

Title:

The high resolution structure of mycobacterium tuberculosis leub (rv2995c)

Structure:

3-isopropylmalate dehydrogenase. Chain: a, b, c, d. Synonym: beta-ipm dehydrogenase, imdh, 3-ipm-dh. Engineered: yes. Other_details: rossman fold

Source:

Mycobacterium tuberculosis. Organism_taxid: 83332. Strain: h37rv. Expressed in: escherichia coli. Expression_system_taxid: 562

Biol. unit:

Dimer (from PDB file)

Resolution:

1.65Å R-factor: 0.213 R-free: 0.241

Authors:

R.K.Singh,G.Kefala,R.Janowski,C.Mueller-Dieckmann,M.S.Weiss,Tb Structural Genomics Consortium (Tbsgc)

Key ref:

R.K.Singh et al. (2005). The high-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis. J Mol Biol, 346, 1. PubMed id: 15663922 DOI: 10.1016/j.jmb.2004.11.059

Date:

03-Jun-04 Release date: 14-Dec-04

Protein chains

P9WKK9  (LEU3_MYCTU) -  3-isopropylmalate dehydrogenase from Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)

Seq: 336 a.a.

Struc: 337 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.1.1.85 - 3-isopropylmalate dehydrogenase.
• Pathway: Leucine Biosynthesis
• Reaction: (2R,3S)-3-isopropylmalate + NAD⁺ = 4-methyl-2-oxopentanoate + CO2 + NADH

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2004.11.059

J Mol Biol 346:1 (2005)

PubMed id: 15663922

The high-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis.

R.K.Singh, G.Kefala, R.Janowski, C.Mueller-Dieckmann, J.P.von Kries, M.S.Weiss.

ABSTRACT

The crystal structure of the enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Mycobacterium tuberculosis (LeuB, Mtb-IPMDH, Rv2995c) without substrate or co-factor was determined at 1.65 A resolution, which is the highest resolution reported for an IPMDH to date. The crystals contain two functional dimers in the asymmetric unit in an arrangement close to a tetramer of D2 symmetry. Despite the absence of a substrate or inhibitor bound to the protein, the structure of the monomer resembles the previously observed closed form of the enzyme more closely than the open form. A comparison with the substrate complex of IPMDH from Thiobacillus ferrooxidans and the co-factor complex of the Thermus thermophilus enzyme revealed a close relationship of the active-site architecture between the various bacterial enzymes. The inhibitor O-isobutenyl oxalylhydroxamate was found to bind to the active site of IPMDH in a mode similar to the substrate isopropylmalate.

Selected figure(s)

Figure 1.
Figure 1. The three-dimensional structure of Mtb-IPMDH. (a) C^a representation of the four subunits of Mtb-IPMDH found in the asymmetric unit. Each subunit is represented by a different colour (A, red; B, green; C, yellow; D, cyan). The six local dyads and the approximate local dyads are indicated. The resemblance of the arrangement to a D[2] tetramer is clearly discernible. (b) Ribbon representation of the structure of the homodimer of Mtb-IPMDH. The two domains in one subunit are identified by different colours, domain 1 is displayed in red, domain 2 in blue. The two different shades of red in domain 1 denote the two sub-domains (see the text). (c) Ribbon representation of the structure of one subunit of Mtb-IPMDH. The secondary structure elements are coloured with a-helices in red and b-strands in yellow. The N and C termini are labelled. The 1.0 kb gene fragment of Rv2995c was amplified by PCR from M. tuberculosis H37Rv genomic DNA with the following oligonucleotides (obtained from MWG Biotech): 5'-AAAATCATGAGCAAACTCGCGATCATTGCCGGTGACGGGATCGGGC-3' and 5'-AAAACTCGAGTTAGAGCGCGGCGGCAATCCGTTCGCCGACA-3' as forward and reverse primers, respectively. In the forward primer, the additional underlined bases were introduced to get the gene in-frame for transcription. Therefore, the gene product obtained contains an extra serine residue at position 2 (codon AGC). The amplified fragment containing 5'-BspHI and 3'-XhoI restriction sites (shown in bold in the primer sequence) was cloned into PCR-Blunt TOPO vector (Invitrogen). The construct was digested with BspHI and XhoI, and the insert fragment was subcloned into the pETM-11 expression vector digested with the restriction enzymes NcoI and XhoI. Both TOPO and the final pETM-11 constructs were sequenced to confirm the cloning of the leuB gene sequence in-frame. Expression of the 35 kDa protein was carried out in E. coli BL21 Star (DE3) pRARE cells. These cells were prepared by transforming BL21 Star (DE3) cells (Invitrogen) with the pRARE plasmid isolated from the Rosetta strain (Novagen). Cells from an overnight pre-culture were grown in LB broth medium containing kanamycin (30 µg/ml) and chloramphenicol (10 µg/ml) at 310 K and 200 rpm. The culture was induced with 0.2 mM isopropyl b-D-thiogalactopyranoside (IPTG) at an A[600] of approximately 0.6 at 293 K. Following induction, the culture was incubated for about 15 hours at 293 K and 200 rpm and then harvested. The cells were stored at 193 K until further processing. One gram of cell pellet was lysed by sonication in 30 ml of buffer A (50 mM Tris (pH 8.0), 200 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, 2 mM b-mercaptoethanol (b-ME)) for 20 minutes in 25 second pulses at 4 °C. The cell debris was pelleted by centrifugation for 30 minutes at 4 °C and 20,000 rpm. The crude lysate was filtered through a 0.2 µm pore size membrane and loaded onto a 5 ml Hi-Trap Chelating HP column (Amersham Biosciences) charged and equilibrated with Ni2+ and buffer A, respectively. In order to remove unbound proteins, the column was first washed with 25 ml of buffer A, then with 25 ml of buffer B (50 mM Tris (pH 8.0), 600 mM NaCl, 10 mM imidazole, 5% (v/v) glycerol, 2 mM b-ME) and then with 25 ml of buffer C (50 mM Tris (pH 8.0), 200 mM NaCl, 50 mM imidazole, 5% (v/v) glycerol, 2 mM b-ME). The protein was eluted by running a linear gradient from 50 mM to 800 mM imidazole (in buffer C) in a total volume of 50 ml. The peak fractions were pooled and dialysed against buffer D (50 mM Tris (pH 8.0), 200 mM NaCl, 5% (v/v) glycerol, 2 mM b-ME). During dialysis overnight at 277 K, the His tag was cleaved off by adding recombinant tobacco etch virus (TEV) protease. The cleaved and dialysed protein mixture was passed through a Ni-NTA column pre-equilibrated with buffer D. The protein in the flow-through was subsequently purified by gel-filtration (Superdex 200 16/60, Amersham Biosciences) using buffer D for both equilibration and elution. The protein eluted with an apparent molecular mass of approximately 70 kDa, which indicated the presence of a homodimer in solution. The peak fractions analysed by SDS-PAGE were pooled, and concentrated to 10 mg/ml. The purity of the protein was estimated by SDS-PAGE and by dynamic light-scattering. The initial crystallization screening was done in Greiner 96-well plates using the sitting-drop, vapour-diffusion method (1 µl of protein and 1 µl of Hampton crystal screens 1 and 2). A few needles were observed in condition #32 of screen #2 (1.6 M (NH[4])[2]SO[4], 0.1 M Hepes (pH 7.5), 0.1 M NaCl) at 293 K. This condition was optimized to 2.0 M (NH[4])[2]SO[4], 0.1 M Hepes (pH 8.0), 0.1 M NaCl. The crystallization experiments using the optimized condition as reservoir solution were performed in 24-well plates using the hanging-drop method with 1.5 µl of protein and 1 µl of reservoir solution at 293 K. Crystals were typically obtained within 48 hours and grew up to 1.0 mm in size. The crystals belong to space group P2[1]2[1]2[1] with unit cell dimensions of a=78.47 Å, b=97.06 Å and c=181.91 Å, and have four molecules in the asymmetric unit. They diffract X-rays to about 1.5 Å resolution. Crystals were treated with cryoprotectant (22% (v/v) glycerol in reservoir solution) and diffraction data were collected at 100 K on the EMBL wiggler beamline BW7B (DESY, Hamburg, Germany) using a MAR345 mm imaging plate detector. Two sweeps of data were collected: a low-resolution sweep (180°) extending to 3.0 Å resolution and a high-resolution sweep (180°) extending to 1.65 Å resolution. The data were indexed and integrated using DENZO23 and scaled using SCALEPACK.23 The redundancy-independent merging R-factor R[r.i.m.] as well as the precision-indicating merging R-factor R[p.i.m.]24 were calculated using the program RMERGE (available from http://www.embl-hamburg.de/ msweiss/projects/msw_qual.html or from M.S.W. upon request). The relevant data processing parameters are given in Table 2. Intensities were converted to

Figure 4.
Figure 4. (a) A representation of the proposed IPM and NAD^+ binding sites in Mtb-IPMDH. (b) A representation of the proposed O-isobutenyl oxalylhydroxamate (O-ibOHA) binding site. All amino acid residues contacting either of the three substances at a distance of less than 4.5 Å are shown. The program GOLD was used for the docking experiments.22 The starting coordinates for IPM and the inhibitor O-ibOHA were generated by superimposing the IPM complex of IPMDH of T. ferrooxidans (PDB entry 1A0510) onto the AB dimer of Mtb-IPMDH. The coordinates of the C3 atom of IPM were defined as the centre of the search sphere. The search was then carried out within a radius of 10 Å, using the default settings of GOLD, including cavity detection and a limit of 50 different dockings.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 346, 1-0) copyright 2005.

Figures were selected by an automated process.