M. tuberculosis N-succinyldiaminopimelate aminotransferase, the enzyme which catalyzes the sixth reaction in the lysine-biosynthesis pathway, has been cloned, expressed, purified and crystallized.
N-Succinyldiaminopimelate aminotransferase from Mycobacterium tuberculosis (DAP-AT; DapC; Rv0858c) has been cloned, heterologously expressed in Escherichia coli, purified using standard chromatographic techniques and crystallized in two related crystal forms. Preliminary diffraction data analysis suggests the presence of a monomer in the asymmetric unit of the tetragonal crystal form and a dimer in the asymmetric unit of the orthorhombic crystal form.
N-succinyldiaminopimelate aminotransferase; DapC; Mycobacterium tuberculosis; Rv0858c
A new series of pyrimidine-requiring mutants of Neurospora has been isolated and all enzymes involved in pyrimidine biosynthesis are represented by at least one mutant. Among these mutants is included a single isolate for a new locus, pyr-6. This mutant is deficient in dihydroorotase (DHOase) and represents the only enzymatic step in orotate synthesis for which no mutant previously had been found. This mutant, which mapped genetically on the right arm of linkage group V, is unlinked to any of the other pyrimidine mutants. The DHOase-deficient mutant is also characterized by an unexpected growth behavior. The pyr-1 locus has been specifically associated with a lack of dihydroorotate dehydrogenase (DHOdehase). Mutants isolated in this series for other pyrimidine loci have been related to previously isolated mutants by allelism, recombination, and accumulation studies.
The N- and C-terminal fragments of alanyl-tRNA synthetase from the archaeon A. fulgidus have been crystallized.
In order to analyze the alanyl-tRNA synthetase from the archaeon Archaeoglobus fulgidus, the N-terminal fragment lacking the dimerization domain and the C-terminal dimerization-domain fragment were each overexpressed in Escherichia coli, purified and crystallized. A 3.7 Å resolution data set was collected for the N-terminal fragment. The crystal belongs to the tetragonal space group P41 or P43, with unit-cell parameters a = b = 101.15, c = 124.24 Å. For the C-terminal fragment, a SeMet MAD data set was collected to 3.2 Å resolution. The crystal belongs to the orthorhombic space group P2221, with unit-cell parameters a = 124.15, b = 131.91, c = 138.68 Å.
AlaRS; tRNa; alanine; editing; dimerization; Archaeoglobus fulgidus
A comparison is made between the structures of dihydroorotase from four different organisms, including B. anthracis dihydroorotase, and reveals substantial variations in the active site, dimer interface and overall tertiary structure.
Dihydroorotase (EC 188.8.131.52) catalyzes the reversible cyclization of N-carbamoyl-l-aspartate to l-dihydroorotate in the third step of the pyrimidine-biosynthesis pathway in Bacillus anthracis. A comparison is made between the structures of dihydroorotase from four different organisms, including B. anthracis dihydroorotase, and reveals substantial variations in the active site, dimer interface and overall tertiary structure. These differences demonstrate the utility of exploring multiple structures of a molecular target as expressed from different organisms and how these differences can be exploited for structure-based drug discovery.
dihydroorotatse; Bacillus anthracis; pyrimidine biosynthesis
The pyrC gene of Escherichia coli K-12, which encodes the pyrimidine biosynthetic enzyme dihydroorotase, was cloned as part of a 1.6-kilobase-pair chromosomal fragment. The nucleotide sequence of this fragment was determined. An open reading frame encoding a 348-amino acid polypeptide (Mr = 38,827) was identified as the pyrC structural gene by comparing the amino acid composition predicted from the DNA sequence with that previously determined for the dihydroorotase subunit. The pyrC promoter was mapped by primer extension of in vivo transcripts. Transcriptional initiation was shown to occur within a region located 36 to 39 base pairs upstream of the pyrC structural gene. Pyrimidine availability appears to affect the use of the minor transcriptional initiation sites. The level of pyrC transcription and dihydroorotase synthesis was coordinately derepressed by pyrimidine limitation, indicating that regulation occurs, at least primarily, at the transcriptional level. Inspection of the pyrC nucleotide sequence indicates that gene expression is not regulated by an attenuation control mechanism similar to that described for the pyrBI operon and the pyrE gene. A possible mechanism of transcriptional control involving a common repressor protein is suggested by the identification of a highly conserved, operatorlike sequence in the promoter regions of pyrC and the other pyrimidine genes (i.e., pyrD and carAB) whose expression is negatively regulated by a cytidine nucleotide effector.
Two C-terminal fragments of the RNA helicase Hera have been crytallized in three crystal forms, one of which was phased by MAD using a single selenium site.
Heat-resistant RNA-dependent ATPase (Hera) from Thermus thermophilus is a DEAD-box RNA helicase. Two constructs encompassing the second RecA-like domain and the C-terminal domain of Hera were overproduced in Escherichia coli and purified to homogeneity. Single crystals of both Hera constructs were obtained in three crystal forms. A tetragonal crystal form belonged to space group P41212, with unit-cell parameters a = 65.5, c = 153.0 Å, and contained one molecule per asymmetric unit. Two orthorhombic forms belonged to space group P212121, with unit-cell parameters a = 62.8, b = 70.9, c = 102.3 Å (form I) and a = 41.6, b = 67.6, c = 183.5 Å (form II). Both orthorhombic forms contained two molecules per asymmetric unit. All crystals diffracted X-rays to beyond 3 Å resolution, but the tetragonal data sets displayed high Wilson B values and high mean |E
2 − 1| values, indicating potential disorder and anisotropy. The tetragonal crystal was phased by MAD using a single selenium site.
Hera; Thermus thermophilus; DEAD-box RNA helicases; ATPases
The transaldolase enzyme from T. acidophilum has been crystallized in two different space groups.
The metabolic enzyme transaldolase from Thermoplasma acidophilum was recombinantly expressed in Escherichia coli and could be crystallized in two polymorphic forms. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 6000 as precipitant. Native data sets for crystal forms 1 and 2 were collected in-house to resolutions of 3.0 and 2.7 Å, respectively. Crystal form 1 belonged to the orthorhombic space group C2221 with five monomers per asymmetric unit and crystal form 2 belonged to the monoclinic space group P21 with ten monomers per asymmetric unit.
Schiff bases; (α/β)8-barrel; fructose 6-phosphate; aldolases; pentose phosphate pathway
The regulation of several enzymes involved in pyrimidine biosynthesis in Neurospora crassa has been studied. Elevation of ATCase (l-aspartate carbamoyltransferase) activity is found in all pyrimidine-requiring mutants when they are starved for uridine. DHOase (dihydroorotase) is an unstable enzyme, and it is impossible to conclude what type of regulation, if any, controls this enzyme. DHOdehase (dihydroorotate dehydrogenase) activity shows a marked elevation in uridine-starved pyr-2 cultures, a mutant blocked late in the pathway. Several mutants blocked early in the pathway show much smaller increases in DHOdehase activity and possible explanations for this are discussed. Differences in the modes of regulation of the pyrimidine biosynthetic pathways in various organisms are compared.
The pathway of pyrimidine biosynthesis in Pseudomonas aeruginosa has been shown to be the same as in other bacteria. Twenty-seven mutants requiring uracil for growth were isolated and the mutant lesions were identified. Mutants lacking either dihydroorotic acid dehydrogenase, orotidine monophosphate pyrophosphorylase, orotidine monophosphate decarboxylase, or aspartic transcarbamylase were isolated; none lacking dihydroorotase were found. By using transduction and conjugation, four genes affecting pyrimidine biosynthetic enzymes have been identified and shown to be unlinked to each other. The linkage of pyrB to met-28 and ilv-2 was shown by contransduction. Repression by uracil alone or by broth could not be demonstrated for any enzymes of this pathway, in contrast to the situation in Escherichia coli and Serratia marcescens. In addition, derepression of these enzymes could not be demonstrated. A low level of feedback inhibition of aspartic transcarbamylase was found to occur. It is suggested that the control of such constitutive biosynthetic enzymes in P. aeruginosa may be related to the comprehensive metabolic activities of this organism.
The enzyme aspartate semialdehyde dehydrogenase from M. tuberculosis has been expressed, purified and crystallized in two different crystal forms.
Aspartate semialdehyde dehydrogenase from Mycobacterium tuberculosis (Asd, ASADH, Rv3708c), which is the second enzyme in the lysine/homoserine-biosynthetic pathways, has been expressed heterologously in Escherichia coli. The enzyme was purified using affinity and gel-filtration chromatographic techniques and crystallized in two different crystal forms. Preliminary diffraction data analysis suggested the presence of up to four monomers in the asymmetric unit of the orthorhombic crystal form A and of one or two monomers in the cubic crystal form B.
aspartate semialdehyde dehydrogenase; Mycobacterium tuberculosis; Rv3708c
Recombinant squid calexcitin has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P212121.
The neuronal protein calexcitin from the long-finned squid Loligo pealei has been expressed in Escherichia coli and purified to homogeneity. Calexcitin is a 22 kDa calcium-binding protein that becomes up-regulated in invertebrates following Pavlovian conditioning and is likely to be involved in signal transduction events associated with learning and memory. Recombinant squid calexcitin has been crystallized using the hanging-drop vapour-diffusion technique in the orthorhombic space group P212121. The unit-cell parameters of a = 46.6, b = 69.2, c = 134.8 Å suggest that the crystals contain two monomers per asymmetric unit and have a solvent content of 49%. This crystal form diffracts X-rays to at least 1.8 Å resolution and yields data of high quality using synchrotron radiation.
calexcitin; calcium-binding proteins
Human ADP-ribosylhydrolase 1, which cleaves the glycosidic bond between ADP-ribose and specific Arg residues in proteins, has been cloned, expressed, purified and crystallized.
Human ADP-ribosylhydrolase 1 (hARH1, ADPRH) cleaves the glycosidic bond of ADP-ribose attached to an Arg residue of a protein. hARH1 has been cloned, expressed heterologously in Escherichia coli, purified and crystallized in complex with K+ and ADP. The orthorhombic crystals contained one monomer per asymmetric unit, exhibited a solvent content of 43% and diffracted X-rays to a resolution of 1.9 Å. A prerequisite for obtaining well diffracting crystals was the performance of X-ray fluorescence analysis on poorly diffracting apo hARH1 crystals, which revealed the presence of trace amounts of K+ in the crystal. Adding K-ADP to the crystallization cocktail then resulted in a crystal of different morphology and with dramatically improved diffraction properties.
ADP-ribosylation; ADP-ribosylhydrolase; ADP-ribosylarginine hydrolase; X-ray fluorescence; ARH1; ADPRH
The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.
Dihydroorotate dehydrogenases (DHODs) are FMN-containing enzymes that catalyze the conversion of dihydroorotate (DHO) to orotate in the de novo synthesis of pyrimidines. During the reaction, a proton is transferred from C5 of DHO to an active site base and the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of the flavin as a hydride. In Class 2 DHODs, a hydrogen-bond network observed in crystal structures has been proposed to deprotonate the C5 atom of DHO. The active site base (Ser175 in the E. coli enzyme) hydrogen bonds to a crystallographic water molecule that sits on a phenylalanine (Phe115 in the E. coli enzyme) and hydrogen-bonds to a threonine (Thr178 in the E. coli enzyme) – residues that are conserved in Class 2 enzymes. The importance of these residues in the oxidation of DHO was investigated using site-directed mutagenesis. Mutating Ser175 to alanine had severe effects on the rate of flavin reduction, slowing it by more than three orders of magnitude. Changing the size and/or hydrophobicity of the residues of the hydrogen-bonding network, Thr178 and Phe115, slowed flavin reduction as much as two orders of magnitude, indicating that the active site base and the hydrogen-bond network work together for efficient deprotonation of DHO.
The crystallization and preliminary X-ray diffraction analysis of sarcosine dimethylglycine methyltransferase from H. halochoris is reported.
Sarcosine dimethylglycine methyltransferase (EC 184.108.40.206) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 Å resolution and the orthorhombic crystal diffracted to 1.8 Å resolution.
sarcosine dimethylglycine methyltransferase; Halorhodospira halochoris; twinning
Mouse peroxiredoxin II was crystallized in an orthorhombic space group and native X-ray diffraction data were collected.
Peroxiredoxin II was cloned from mouse B cells into pCold 1 expression vector and produced as a His-tagged recombinant protein in Escherichia coli. A ring form was isolated by gel filtration. A crystal obtained by the sitting-drop vapour-diffusion method diffracted to 1.77 Å resolution at 100 K. The crystal belonged to space group P21212, with unit-cell parameters a = 117.4, b = 133.9, c = 139.1 Å. The asymmetric unit is expected to contain six dimers of peroxiredoxin II, with a corresponding solvent content of 39.3%. Peaks in the native Patterson function together with pseudo-systematic absences suggested that the crystals suffered from severe translational pseudosymmetry.
Crystallization and preliminary X-ray diffraction studies are reported for a novel Kunitz-type protease inhibitor from B. bauhinioides which contains no disulfide bridges.
A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 Å resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P212121, with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 Å. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (V
M = 2.5 Å3 Da−1). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 Å resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.
Kunitz-type kallikrein inhibitors; disulfide bridges; Bauhinia bauhinioides
The sulfide:quinone oxidoreductase from A. ferrooxidans ATCC 23270 was overexpressed in E. coli and purified. Crystallization and preliminarily X-ray crystallographic analysis were performed for the recombinant enzyme.
The gene product of open reading frame AFE_1293 from Acidithiobacillus ferrooxidans ATCC 23270 is annotated as encoding a sulfide:quinone oxidoreductase, an enzyme that catalyses electron transfer from sulfide to quinone. Following overexpression in Escherichia coli, the enzyme was purified and crystallized using the hanging-drop vapour-diffusion method. The native crystals belonged to the tetragonal space group P42212, with unit-cell parameters a = b = 131.7, c = 208.8 Å, and diffracted to 2.3 Å resolution. Preliminary crystallographic analysis indicated the presence of a dimer in the asymmetric unit, with an extreme value of the Matthews coefficient (V
M) of 4.53 Å3 Da−1 and a solvent content of 72.9%.
sulfide:quinone reductase oxidoreductases
Single crystals of recombinant S-adenosyl-l-homocysteine hydrolase from L. luteus in complex with adenosine diffract X-rays to 1.17 Å resolution at 100 K. The crystals are tetragonal, space group P43212, and contain one copy of the dimeric enzyme in the asymmetric unit.
By degrading S-adenosyl-l-homocysteine, which is a byproduct of S-adenosyl-l-methionine-dependent methylation reactions, S-adenosyl-l-homocysteine hydrolase (SAHase) acts as a regulator of cellular methylation processes. S-Adenosyl-l-homocysteine hydrolase from the leguminose plant yellow lupin (Lupinus luteus), LlSAHase, which is composed of 485 amino acids and has a molecular weight of 55 kDa, has been cloned, expressed in Escherichia coli and purified. Crystals of LlSAHase in complex with adenosine were obtained by the hanging-drop vapour-diffusion method using 20%(w/v) PEG 4000 and 10%(v/v) 2-propanol as precipitants in 0.1 M Tris–HCl buffer pH 8.0. The crystals were tetragonal, space group P43212, with unit-cell parameters a = 122.4, c = 126.5 Å and contained two protein molecules in the asymmetric unit, corresponding to the functional dimeric form of the enzyme. Atomic resolution (1.17 Å) X-ray diffraction data have been collected using synchrotron radiation.
S-adenosyl-l-homocysteine hydrolase; Lupinus luteus
The preliminary crystallographic analysis of the N-terminal domain of FILIA is described in this paper. FILIA is a component of subcortical maternal complex, which plays critical roles in embryogenesis.
FILIA is a component of the subcortical maternal complex that is essential for early stage embryogenesis. Its 6×His-tagged N-terminal domain was expressed in Escherichia coli and purified to homogeneity. Two types of crystals formed under different crystallization conditions during screening. Orthorhombic crystals appeared in a solution containing 1.4 M ammonium sulfate, 0.1 M Tris pH 8.2 and 12% glycerol, while tetragonal crystals were obtained using 15% PEG 4000 mixed with 0.1 M HEPES pH 7.5 and 15% 2-propanol. High-quality diffraction data were collected from the two crystal forms to resolutions of 1.8 and 2.2 Å, respectively, using synchrotron radiation. The Matthews coefficients indicated that the P212121 and P41212 crystals contained two molecules and one molecule per asymmetric unit, respectively. A selenomethionine-substituted sample failed to crystallize under the native conditions, but another orthorhombic crystal form was obtained under different conditions and anomalous diffraction data were collected.
The crystallization of E. coli maltoporin in a new crystal form that diffracts to high resolution is reported.
Maltoporin is an outer-membrane protein that forms a β-barrel composed of three monomers and ensures the transport of maltose and maltodextrin in Gram-negative bacteria. Previously, the crystallization of Escherichia coli or Salmonella typhimurium maltoporin has been achieved in the presence of a mixture of the detergents β-decylmaltoside and dodecyl nonaoxyethylene. These crystals all belonged to the orthorhombic space group C2221 and gave rise to several structures of maltoporin in complex with different carbohydrates determined at resolutions between 3.2 and 2.4 Å. Here, the crystallization of E. coli maltoporin in a new crystal form is reported; the crystals belonged to the trigonal R3 space group and diffracted to 1.9 Å resolution. These crystals were obtained using n-dodecyl-β-d-maltoside as a detergent. Crystals with a lens or pyramidal morphology could be obtained using sitting or hanging drops, respectively, and despite their very different shapes they presented the same space group and very similar unit-cell parameters.
outer-membrane proteins; maltoporin; carbohydrates
The response regulator DesR from S. pneumoniae was cloned, expressed, purified and crystallized. A complete data set was collected to 1.7 Å resolution.
The response regulator DesR, which activates the transcription of the des gene by binding to a regulatory region, is essential for controlling the fluidity of membrane phospholipids. DesR from Streptococcus pneumoniae was overexpressed in Escherichia coli. The protein was purified and crystallized for structural analysis. Diffraction data were collected to 1.7 Å resolution using synchrotron radiation and the crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 46.91, b = 71.38, c = 117.73 Å. Assuming the presence of a dimer in the asymmetric unit, this corresponds to a V
M of 2.21 Å3 Da−1.
two-component systems; fatty-acid desaturation; response regulators
An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme.
The 34 kDa main proteinase (Mpro) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV Mpro is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P21212 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.
protease; crystallographic dimer; SARS coronavirus
Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively.
The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P212121 and tetragonal P41212) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*65–224 structure was determined by molecular replacement.
rotaviruses; VP8*; sialic acid
Dihydroorotate dehydrogenases (DHODs) catalyze the only redox step in de novo pyrimidine biosynthesis, the oxidation of dihydroorotate (DHO) to orotate (OA). During the reaction, the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of an enzyme-bound FMN prosthetic group as a hydride and an active site base (Ser175 in the Class 2 DHOD from E. coli) deprotonates C5 of DHO. Aside from the identity of the active site base, the pyrimidine binding site of all DHODs is nearly identical. Several strictly conserved residues (four asparagines and either a serine or threonine) make extensive hydrogen-bonds to the pyrimidine). The roles these conserved residues play in DHO oxidation is unknown. Site-directed mutagenesis was used to investigate the role of each residue during DHO oxidation. The effects of each mutation on substrate and product binding, as well as the effect on the rate constant of the chemical step were determined. The effects of the mutations ranged from negligible to severe. Some of the residues are very important for chemistry, while others were important for binding. Mutation of residues capable of stabilizing reaction intermediates resulted in large decreases in the rate constant of the chemical step, suggesting these residues are quite important for stabilizing charge build-up in the active site. This finding is consistent with previous results that Class 2 DHODs use a stepwise mechanism for DHO oxidation.