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1.  Bacterial variations on the methionine salvage pathway 
BMC Microbiology  2004;4:9.
Background
The thiomethyl group of S-adenosylmethionine is often recycled as methionine from methylthioadenosine. The corresponding pathway has been unravelled in Bacillus subtilis. However methylthioadenosine is subjected to alternative degradative pathways depending on the organism.
Results
This work uses genome in silico analysis to propose methionine salvage pathways for Klebsiella pneumoniae, Leptospira interrogans, Thermoanaerobacter tengcongensis and Xylella fastidiosa. Experiments performed with mutants of B. subtilis and Pseudomonas aeruginosa substantiate the hypotheses proposed. The enzymes that catalyze the reactions are recruited from a variety of origins. The first, ubiquitous, enzyme of the pathway, MtnA (methylthioribose-1-phosphate isomerase), belongs to a family of proteins related to eukaryotic intiation factor 2B alpha. mtnB codes for a methylthioribulose-1-phosphate dehydratase. Two reactions follow, that of an enolase and that of a phosphatase. While in B. subtilis this is performed by two distinct polypeptides, in the other organisms analyzed here an enolase-phosphatase yields 1,2-dihydroxy-3-keto-5-methylthiopentene. In the presence of dioxygen an aci-reductone dioxygenase yields the immediate precursor of methionine, ketomethylthiobutyrate. Under some conditions this enzyme produces carbon monoxide in B. subtilis, suggesting a route for a new gaseous mediator in bacteria. Ketomethylthiobutyrate is finally transaminated by an aminotransferase that exists usually as a broad specificity enzyme (often able to transaminate aromatic aminoacid keto-acid precursors or histidinol-phosphate).
Conclusion
A functional methionine salvage pathway was experimentally demonstrated, for the first time, in P. aeruginosa. Apparently, methionine salvage pathways are frequent in Bacteria (and in Eukarya), with recruitment of different polypeptides to perform the needed reactions (an ancestor of a translation initiation factor and RuBisCO, as an enolase, in some Firmicutes). Many are highly dependent on the presence of oxygen, suggesting that the ecological niche may play an important role for the existence and/or metabolic steps of the pathway, even in phylogenetically related bacteria. Further work is needed to uncover the corresponding steps when dioxygen is scarce or absent (this is important to explore the presence of the pathway in Archaea). The thermophile T. tengcongensis, that thrives in the absence of oxygen, appears to possess the pathway. It will be an interesting link to uncover the missing reactions in anaerobic environments.
doi:10.1186/1471-2180-4-9
PMCID: PMC395828  PMID: 15102328
2.  Structure of MRDI Explains its Dual Function as a Metabolic Enzyme and a Mediator of Cell Invasion 
Biochemistry  2013;52(33):10.1021/bi400556e.
Metastatic melanoma is among the most intractable cancers to treat, where patients show resistance to therapy and limited survival time. A critical step in the development of metastatic melanoma is the acquisition of invasion and transition from thin to thick tumors on the skin, followed by invasion to lymph nodes. Prior studies have shown that metastatic melanoma is associated with dysregulation of RhoA and enhanced expression of a protein named “mediator of RhoA-dependent invasion (MRDI)”. Importantly, MRDI is a “moonlighting” enzyme, with two distinct functions in melanoma cells. First, MRDI acts as a methylthioribose-1-phosphate (MTR-1-P) isomerase, catalyzing a critical step in methionine salvage. Second, MRDI promotes and is necessary for melanoma cell invasion, independent of its catalytic activity. Here, we demonstrate that MtnA, a bacterial MTR-1-P isomerase, rescues the methionine salvage function of MRDI, but is unable to rescue its role in invasion. We then solve the crystal structure of MRDI to a resolution of 2.5 Å, in order to identify structural elements important for its invasion activity. We present this structure and its comparison with other MTR-1-P isomerases, and identify mutations within a region separate from the MTR-1-P binding site which interfere with invasion. Thus, structural elements in MRDI distal from the MTR-1-P catalytic site are responsible for the invasion phenotype.
doi:10.1021/bi400556e
PMCID: PMC3831603  PMID: 23859498
Methionine Salvage; Melanoma; Invasion; Moonlighting Enzymes; Methylthioribose-1-phosphate Isomerase
3.  Functional Identification of APIP as Human mtnB, a Key Enzyme in the Methionine Salvage Pathway 
PLoS ONE  2012;7(12):e52877.
The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.
doi:10.1371/journal.pone.0052877
PMCID: PMC3532061  PMID: 23285211
4.  MtnK, methylthioribose kinase, is a starvation-induced protein in Bacillus subtilis 
BMC Microbiology  2001;1:15.
Background
Methylthioadenosine, the main by-product of spermidine synthesis, is degraded in Bacillus subtilis as adenine and methylthioribose. The latter is an excellent sulfur source and the precursor of quorum-sensing signalling molecules. Nothing was known about methylthioribose recycling in this organism.
Results
Using trifluoromethylthioribose as a toxic analog to select for resistant mutants, we demonstrate that methylthioribose is first phosphorylated by MtnK, methylthioribose kinase, the product of gene mtnK (formerly ykrT), expressed as an operon with mtnS (formerly ykrS) in an abundant transcript with a S-box leader sequence. Although participating in methylthioribose recycling, the function of mtnS remained elusive. We also show that MtnK synthesis is boosted under starvation condition, in the following decreasing order: carbon-, sulfur- and nitrogen-starvation. We finally show that this enzyme is part of the family Pfam 01633 (choline kinases) which belongs to a large cluster of orthologs comprizing antibiotic aminoglycoside kinases and protein serine/threonine kinases.
Conclusions
The first step of methylthioribose recycling is phosphoryltaion by MTR kinase, coded by the mtnK (formerly ykrT) gene. Analysis of the neighbourhood of mtnK demonstrates that genes located in its immediate vicinity (now named mtnUVWXYZ, formerly ykrUVWXYZ) are also required for methylthioribose recycling.
PMCID: PMC55331  PMID: 11545674
5.  Crystallization and preliminary X-ray analysis of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from Bacillus subtilis  
Crystals of the 45.1 kDa functional form of 2,3-diketo-5-methylthiopentyl-1-phosphate enolase from B. subtilis diffracted to 2.30 Å resolution.
2,3-Diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) from Bacillus subtilis was crystallized using the hanging-drop vapour-diffusion method. Crystals grew using PEG 3350 as the precipitant at 293 K. The crystals diffracted to 2.3 Å resolution at 100 K using synchrotron radiation and were found to belong to the monoclinic space group P21, with unit-cell parameters a = 79.3, b = 91.5, c = 107.0 Å, β = 90.8°. The asymmetric unit contained four molecules of DK-MTP-1P enolase, with a V M value of 2.2 Å3 Da−1 and a solvent content of 43%.
doi:10.1107/S174430910804311X
PMCID: PMC2635871  PMID: 19194007
methionine-salvage pathway; Bacillus subtilis; RuBisCO; RuBisCO-like proteins; 2,3-diketo-5-methylthiopentyl-1-phosphate enolase
6.  MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of Tetrahymena thermophila 
PLoS ONE  2013;8(7):e67385.
To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and 1H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.
doi:10.1371/journal.pone.0067385
PMCID: PMC3698126  PMID: 23840871
7.  The methionine salvage pathway in Bacillus subtilis 
BMC Microbiology  2002;2:8.
Background
Polyamine synthesis produces methylthioadenosine, which has to be disposed of. The cell recycles it into methionine through methylthioribose (MTR). Very little was known about MTR recycling for methionine salvage in Bacillus subtilis.
Results
Using in silico genome analysis and transposon mutagenesis in B. subtilis we have experimentally uncovered the major steps of the dioxygen-dependent methionine salvage pathway, which, although similar to that found in Klebsiella pneumoniae, recruited for its implementation some entirely different proteins. The promoters of the genes have been identified by primer extension, and gene expression was analyzed by Northern blotting and lacZ reporter gene expression. Among the most remarkable discoveries in this pathway is the role of an analog of ribulose diphosphate carboxylase (Rubisco, the plant enzyme used in the Calvin cycle which recovers carbon dioxide from the atmosphere) as a major step in MTR recycling.
Conclusions
A complete methionine salvage pathway exists in B. subtilis. This pathway is chemically similar to that in K. pneumoniae, but recruited different proteins to this purpose. In particular, a paralogue or Rubisco, MtnW, is used at one of the steps in the pathway. A major observation is that in the absence of MtnW, MTR becomes extremely toxic to the cell, opening an unexpected target for new antimicrobial drugs. In addition to methionine salvage, this pathway protects B. subtilis against dioxygen produced by its natural biotope, the surface of leaves (phylloplane).
doi:10.1186/1471-2180-2-8
PMCID: PMC113757  PMID: 12022921
8.  Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage ϕ29 
ϕ29 bacteriophage scaffolding protein (gp7) has been overproduced in E. coli, purified, crystallized and characterized by X-ray diffraction. Two distinct crystal forms were obtained and a diffraction data set was collected to 1.8 Å resolution.
The Bacillus subtilis bacteriophage ϕ29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P41212, with unit-cell parameters a = b = 77.13, c = 37.12 Å. Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 Å. Complete data sets have been collected to 1.78 and 1.80 Å for forms I and II, respectively, at 100 K using Cu Kα X-rays from a rotating-anode generator. Calculation of a V M value of 2.46 Å3 Da−1 for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a V M of 4.80 Å3 Da−1 with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.
doi:10.1107/S1744309105008511
PMCID: PMC1952437  PMID: 16511059
scaffolding protein; bacteriophage ϕ29
9.  Purification, crystallization and preliminary X-ray crystallographic study of the l-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8 
The crystallization and preliminary X-ray diffraction analysis of the l-fuculose-1-phosphate aldolase (FucA) from T. thermophilus HB8. Native diffraction data set was collected to a resolution of 1.9 Å.
Fuculose phosphate aldolase catalyzes the reversible cleavage of l-fuculose-1-­phosphate to dihydroxyacetone phosphate and l-lactaldehyde. The protein from Thermus thermophilus HB8 is a biological tetramer with a subunit molecular weight of 21 591 Da. Purified FucA has been crystallized using sitting-drop vapour-diffusion and microbatch techniques at 293 K. The crystals belong to space group P4, with unit-cell parameters a = b = 100.94, c = 45.87 Å. The presence of a dimer of the enzyme in the asymmetric unit was estimated to give a Matthews coefficient (V M) of 2.7 Å3 Da−1 and a solvent content of 54.2%(v/v). Three-wavelength diffraction MAD data were collected to 2.3 Å from zinc-containing crystals. Native diffraction data to 1.9 Å resolution have been collected using synchrotron radiation at SPring-8.
doi:10.1107/S1744309105036766
PMCID: PMC1978142  PMID: 16511238
fuculose phosphate aldolase; class II aldolases; Thermus thermophilus HB8
10.  Crystallization and preliminary X-ray crystallographic analysis of human Apaf-1-interacting protein 
Apaf-1-interacting protein (APIP) inhibits cell death involving caspase-1 and caspase-9. APIP was crystallized and X-ray diffraction data were collected to 2.40 Å resolution.
Apaf-1-interacting protein (APIP) is known to inhibit two different types of cell death: caspase-1-dependent pyroptosis and caspase-9-dependent apoptosis. APIP is also involved in the methionine-salvage pathway, where it is called 5-­methylthioribulose-1-phosphate dehydratase (MtnB). The enzyme activity seems to be essential for inhibition of pyroptosis by APIP, but not for inhibition of apoptosis. In this study, human APIP was overproduced in Escherichia coli, purified and crystallized. An X-ray diffraction data set was collected to 2.40 Å resolution and the crystals belonged to space group C2221, with unit-cell parameters a = 106.61, b = 107.50, c = 189.76 Å. Given that four APIP molecules exist in the asymmetric unit, the Matthews coefficient is 2.70 Å3 Da−1 and the corresponding solvent content is 54.4%.
doi:10.1107/S1744309112042832
PMCID: PMC3509978  PMID: 23192037
Apaf-1; APIP; MtnB
11.  Expression, purification, crystallization and preliminary X-ray analysis of ribitol-5-phosphate cytidylyltransferase from Bacillus subtilis  
The B. subtilis ribitol-5-phosphate cytidylyltransferase enzyme TarI was crystallized; determination of its structure will lead to structural and functional insight into the biosynthesis of wall teichoic acids.
TarI is a ribitol-5-phosphate cytidylyltransferase that catalyzes the formation of CDP-ribitol, which is involved in the biosynthesis of wall teichoic acids, from CTP and ribitol 5-phosphate. TarI from Bacillus subtilis (BsTarI) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.78 Å and belonged to the monoclinic space group C2, with unit-cell parameters a = 103.74, b = 60.97, c = 91.80 Å, β = 113.48°. The initial structural model indicated that the crystals of BsTarI contained a dimer in the asymmetric unit.
doi:10.1107/S1744309112035142
PMCID: PMC3497978  PMID: 23027746
ribitol-5-phosphate cytidylyltransferase; wall teichoic acids; Bacillus subtilis; TarI
12.  Overproduction, crystallization and preliminary X-­ray analysis of the putative l-ascorbate-6-phosphate lactonase UlaG from Escherichia coli  
UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in E. coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized in a monoclinic space group. Crystals were obtained by the sitting-drop vapour-diffusion method at 293 K. A data set diffracting to 3 Å resolution was collected from a single crystal at 100 K.
UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å3 Da−1 and a solvent content of 43%.
doi:10.1107/S1744309107065256
PMCID: PMC2373999  PMID: 18097099
UlaG; l-ascorbate metabolism; enterobacterial metabolism
13.  Crystallization and preliminary X-ray crystallographic analysis of l-rhamnose isomerase with a novel high thermostability from Bacillus halodurans  
l-Rhamnose isomerase (l-RhI) from B. halodurans has been purified and crystallized. The crystals of l-RhI belonged to the monoclinic space group P21, with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°, and diffracted to 2.5 Å resolution.
l-Rhamnose isomerases catalyze isomerization between l-rhamnose (6-deoxy-l-mannose) and l-rhamnulose (6-deoxy-l-fructose), which is the first step in rhamnose catabolism. l-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P21, with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 Å, β = 116.0°. Diffraction data were collected to 2.5 Å resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V M of 3.0 Å3 Da−1 and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method.
doi:10.1107/S174430911001256X
PMCID: PMC2882768  PMID: 20516598
Bacillus halodurans; rhamnose isomerase; thermostability
14.  Crystallization and preliminary X-ray analysis of AzoR (azoreductase) from Escherichia coli  
The crystallization and preliminary X-ray analysis of AzoR (azoreductase) have been performed.
AzoR (azoreductase), an FMN-dependent NADH-azo compound oxidoreductase from Escherichia coli, has been crystallized in the presence of FMN by the sitting-drop vapour-diffusion method using 2-propanol as a precipitant. AzoR catalyzes the reductive cleavage of azo groups. The crystals were found to diffract X-rays to beyond 1.8 Å resolution using a synchrotron-radiation source. The crystals belonged to the tetragonal space group P42212, with unit-cell parameters a = b = 92.2, c = 51.9 Å. The crystals are expected to contain one subunit of the homodimer in the asymmetric unit (V M = 2.6 Å3 Da−1) and to have a solvent content of 51.6%. Data sets were also collected from heavy-atom derivatives for use in phasing. As a result, crystals soaked in a solution containing K2PtCl4 for 23 d were found to be reasonably isomorphous to the native crystals and the presence of Pt atoms could be confirmed. The data sets from the native crystals and the K2PtCl4-derivatized crystals are being evaluated for use in structure determination by single isomorphous replacement with anomalous scattering.
doi:10.1107/S1744309105007918
PMCID: PMC1952434  PMID: 16511052
azoreductases
15.  Crystallisation of Wild-Type and Variant Forms of a Recombinant Plant Enzyme β-d-Glucan Glucohydrolase from Barley (Hordeum vulgare L.) and Preliminary X-ray Analysis 
Wild-type and variant crystals of a recombinant enzyme β-d-glucan glucohydrolase from barley (Hordeum vulgare L.) were obtained by macroseeding and cross-seeding with microcrystals obtained from native plant protein. Crystals grew to dimensions of up to 500 × 250 × 375 μm at 277 K in the hanging-drops by vapour-diffusion. Further, the conditions are described that yielded the wild-type crystals with dimensions of 80 × 40 × 60 μm by self-nucleation vapour-diffusion in sitting-drops at 281 K. The wild-type and recombinant crystals prepared by seeding techniques achived full size within 5–14 days, while the wild-type crystals grown by self-nucleation appeared after 30 days and reached their maximum size after another two months. Both the wild-type and recombinant variant crystals, the latter altered in the key catalytic and substrate-binding residues Glu220, Trp434 and Arg158/Glu161 belonged to the P43212 tetragonal space group, i.e., the space group of the native microcrystals was retained in the newly grown recombinant crystals. The crystals diffracted beyond 1.57–1.95 Å and the cell dimensions were between a = b = 99.2–100.8 Å and c = 183.2–183.6 Å. With one molecule in the asymmetric unit, the calculated Matthews coefficients were between 3.4–3.5 Å3·Da−1 and the solvent contents varied between 63.4% and 64.5%. The macroseeding and cross-seeding techniques are advantageous, where a limited amount of variant proteins precludes screening of crystallisation conditions, or where variant proteins could not be crystallized.
doi:10.3390/ijms11072759
PMCID: PMC2920565  PMID: 20717535
macro- and cross-seeding; wild-type and mutant protein; X-ray diffraction
16.  Expression, purification, crystallization and preliminary X-ray analysis of the KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 
The KaiC-like protein PH0187 from the hyperthermophilic archaeon P. horikoshii OT3 was expressed, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal of PH0187 diffracted X-rays to 2.75 Å resolution.
KaiC is the central protein in the circadian rhythm in cyanobacteria. The 28 kDa KaiC-like protein PH0187 from the hyperthermophilic archaeon Pyrococcus horikoshii was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of PH0187 were obtained using a reservoir solution consisting of 1.0 M ammonium phosphate monobasic and 0.1 M sodium citrate tribasic pH 5.3 (the final pH value of the reservoir solution was 4.8) and diffracted X-rays to 2.75 Å resolution. The crystal of PH0187 belonged to space group P6322, with unit-cell parameters a = b = 239.1, c = 106.5 Å. The crystal contained four PH0187 molecules in the asymmetric unit.
doi:10.1107/S1744309110048426
PMCID: PMC3079995  PMID: 21206047
PH0187; KaiC; circadian rhythm
17.  Overexpression, crystallization and preliminary X-­ray analysis of xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from Bifidobacterium breve  
Xylulose-5-phosphate/fructose-6-phosphate phosphoketolase from B. breve was overexpressed and crystallized. The crystals belonged to the tetragonal space group I422 and diffracted to beyond 1.7 Å resolution.
The xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene from Bifidobacterium breve was cloned and overexpressed in Escherichia coli. The enzyme was purified to homogeneity and crystallized by the sitting-drop vapour-diffusion method. Crystals were obtained at 293 K using 0.05 mM thiamine diphosphate, 0.25 mM MgCl2, 24%(w/v) PEG 6000 and 0.1 M Bicine pH 9.0. The crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 174.8, c = 163.8 Å, and diffracted to beyond 1.7 Å resolution.
doi:10.1107/S1744309110023845
PMCID: PMC2917298  PMID: 20693675
xylulose-5-phosphate/fructose-6-phosphate phosphoketolase; Bifidobacterium breve; bifid shunt; heterofermentative lactic acid bacteria
18.  Expression, crystallization and preliminary crystallographic analysis of the PAS domain of RsbP, a stress-response phosphatase from Bacillus subtilis  
The PAS domain of RsbP, a stress-response protein from B. subtilis, was crystallized using the sitting-drop vapour-diffusion method. The crystals belonged to space group P21 and diffraction data were collected to a resolution of 1.6 Å.
RsbP, a regulator of RNA polymerase σB activity in Bacillus subtilis, is a phosphatase containing a Per–Arnt–Sim (PAS) domain in its N-terminal region that is expected to sense energy stresses such as carbon, phosphate or oxygen starvation. Energy-stress signals are transmitted to the PAS domain and activate the C-terminal phosphatase domain of RsbP, leading to activation of the downstream anti-anti-σB factor RsbV. Finally, the general stress response is induced to protect the cells against further stresses. The recombinant PAS domain of RsbP was crystallized by the sitting-drop vapour-diffusion technique using 40% PEG 400 as a precipitant. The crystals belonged to space group P21, with unit-cell parameters a = 55.2, b = 71.7, c = 60.2 Å, β = 92.1°. Diffraction data were collected to a resolution of 1.6 Å.
doi:10.1107/S1744309109014158
PMCID: PMC2688409  PMID: 19478430
σB; stress response; energy stress; phosphatase; PAS domain
19.  Cloning, purification and preliminary crystallographic analysis of a putative pyridoxal kinase from Bacillus subtilis  
A putative pyridoxal kinase from B. subtilis has been cloned, overexpressed, purified and crystallized and data have been collected to 2.8 Å resolution.
Pyridoxal kinases (PdxK) are able to catalyse the phosphorylation of three vitamin B6 precursors, pyridoxal, pyridoxine and pyridoxamine, to their 5′-­phosphates and play an important role in the vitamin B6 salvage pathway. Recently, the thiD gene of Bacillus subtilis was found to encode an enzyme which has the activity expected of a pyridoxal kinase despite its previous assignment as an HMPP kinase owing to higher sequence similarity. As such, this enzyme would appear to represent a new class of ‘HMPP kinase-like’ pyridoxal kinases. B. subtilis thiD has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a binary complex with ADP and Mg2+. X-ray diffraction data have been collected from crystals to 2.8 Å resolution at 100 K. The crystals belong to a primitive tetragonal system, point group 422, and analysis of the systematic absences suggest that they belong to one of the enantiomorphic pair of space groups P41212 or P43212. Consideration of the space-group symmetry and unit-cell parameters (a = b = 102.9, c = 252.6 Å, α = β = γ = 90°) suggest that the crystals contain between three and six molecules in the asymmetric unit. A full structure determination is under way to provide insights into aspects of the enzyme mechanism and substrate specificity.
doi:10.1107/S1744309106035779
PMCID: PMC2225197  PMID: 17012797
thiD; PdxK; HMPP kinase; pyridoxal kinase; ribokinase superfamily
20.  Overexpression, crystallization and preliminary X-­ray crystallographic analysis of d-ribose-5-phosphate isomerase from Clostridium thermocellum  
Clostridium thermocellum d-­ribose-5-phosphate isomerase has been purified and crystallized in order to determine its three-dimensional structure and thus to elucidate its enzymatic reaction mechanism and understand its substrate specificity.
Rare sugars are used for many industrial and medical purposes and are produced by the interconversion between aldoses and ketoses catalyzed by sugar and sugar-phosphate isomerases. Recently, Clostridium thermocellum d-­ribose-5-phosphate isomerase (CTRPI), an aldose–ketose isomerase, was cloned in order to synthesize d-allose and its substrate specificity was further characterized for industrial usage. CTRPI has a novel substrate specificity that differs from those of other isomerases, which have broad substrate specificities. CTRPI prefers aldose substrates such as l-talose, d-ribose and d-allose. CTRPI was purified and crystallized in order to determine its three-dimensional structure and thus to elucidate its enzymatic reaction mechanism and understand its substrate specificity. The crystal belonged to the trigonal space group P3221, with unit-cell parameters a = b = 69.5, c = 154.4 Å, and diffracted to 1.9 Å resolution. According to Matthews coefficient calculations, the crystallo­graphic structure consists of a dimer in the asymmetric unit, with a V M of 3.2 Å3 Da−1 and a solvent content of 61.7%.
doi:10.1107/S1744309109038093
PMCID: PMC2777044  PMID: 19923736
d-ribose phosphate isomerases; CTRPI; isomerization
21.  Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the α-crystallin family 
XAC1151, a small heat-shock protein from X. axonopodis pv. citri belonging to the α-crystallin family, was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source.
The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 Å resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 Å. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.
doi:10.1107/S174430910601219X
PMCID: PMC2219972  PMID: 16682772
XAC1151; small heat-shock proteins; α-crystallins; Xanthomonas axonopodis
22.  Crystallization and preliminary crystallographic study of the phosphoglucose isomerase from Bacillus subtilis  
Phosphoglucose isomerase from B. subtilis has been purified and crystallized. The diffraction quality of the crystal was improved by using a flash-annealing technique and 1.9 Å resolution in-house X-ray data were collected.
Crystallization and preliminary crystallographic analysis of the phospho­glucose isomerase from a Bacillus subtilis native strain were carried out. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 145.7, b = 136.0, c = 109.1 Å, β = 119.4°. The diffraction quality of the crystal was significantly improved from 2.4 Å to greater than 1.9 Å resolution by using the in situ flash-annealing method. A 98% complete data set with an overall R merge of 4.6% was collected using an R-AXIS IV++ image-plate system and a copper rotating-anode X-ray generator. The crystals contained four molecules per asymmetric unit and the predicted solvent content and the Matthews coefficient (V M) were 46.8% and 2.3 Å3 Da−1, respectively. Structure determination by the molecular-replacement method provided a reasonable solution for model building.
doi:10.1107/S1744309108037718
PMCID: PMC2593699  PMID: 19052382
flash-annealing; thermolability; phosphoglucose isomerase
23.  Crystallization and preliminary X-ray diffraction analysis of the complex of Kunitz-type tamarind trypsin inhibitor and porcine pancreatic trypsin 
A complex of tamarind trypsin inhibitor with porcine trypsin was crystallized and analyzed by X-ray diffraction.
The complex of Tamarindus indica Kunitz-type trypsin inhibitor and porcine trypsin has been crystallized by the sitting-drop vapour-diffusion method using ammonium acetate as precipitant and sodium acetate as buffer. The homogeneity of complex formation was checked by size-exclusion chromatography and further confirmed by reducing SDS–PAGE. The crystals diffracted to 2.0 Å resolution and belonged to the tetragonal space group P41, with unit-cell parameters a = b = 57.1, c = 120.1 Å. Preliminary X-ray diffraction analysis indicated the presence of one unit of inhibitor–trypsin complex per asymmetric unit, with a solvent content of 45%.
doi:10.1107/S1744309109041694
PMCID: PMC2777053  PMID: 19923745
tamarind trypsin inhibitor; porcine pancreatic trypsin; Kunitz-type inhibitors
24.  Crystallization and preliminary X-ray analysis of the complex between a Bacillus subtilis α/β-type small acid-soluble spore protein and DNA 
An α/β-type small, acid-soluble spore protein (SASP) from Bacillus subtilis, a major source of DNA protection against damaging effects in spores, was crystallized in a functionally relevant complex with a double-stranded DNA. This report provides insights into initial characterization of the complex and its structure elucidation.
An engineered variant of an α/β-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281 K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4 Å resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6122 or P6522, with unit-cell parameters a = b = 87.0, c = 145.4 Å, α = β = 90.0, γ = 120.0°. Diffraction data were 96.6% complete to 2.4 Å resolution, with an R sym of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress.
doi:10.1107/S1744309107022750
PMCID: PMC2335083  PMID: 17554173
small acid-soluble spore protein; spore resistance; DNA; Bacillus subtilis
25.  Structure of Arabidopsis thaliana 5-methylthioribose kinase reveals a more occluded active site than its bacterial homolog 
Background
Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics.
Results
The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATPγS and MTR has been determined at 1.9 Å resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATPγS analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR.
Conclusion
The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.
doi:10.1186/1472-6807-7-70
PMCID: PMC2194712  PMID: 17961230

Results 1-25 (196769)