Octavian Bârzu

Decreased Capacity of Recombinant 45/47-kDa Molecules (Apa) of Mycobacterium tuberculosis to Stimulate T Lymphocyte Responses Related to Changes in Their Mannosylation Pattern*

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitrosensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed inEscherichia coli was nearly unable to elicit DTH reactionsin vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.

Structures of Escherichia coli CMP kinase alone and in complex with CDP: a new fold of the nucleoside monophosphate binding domain and insights into cytosine nucleotide specificity

BACKGROUND . Nucleoside monophosphate kinases (NMP kinases) catalyze the reversible transfer of a phosphoryl group from a nucleoside triphosphate to a nucleoside monophosphate. Among them, cytidine monophosphate kinase from Escherichia coli has a striking particularity: it is specific for CMP, whereas in eukaryotes a unique UMP/CMP kinase phosphorylates both CMP and UMP with similar efficiency. RESULTS . The crystal structure of the CMP kinase apoenzyme from E. coli was solved by single isomorphous replacement and refined at 1.75 A resolution. The structure of the enzyme in complex with CDP was determined at 2.0 A resolution. Like other NMP kinases, the protein contains a central parallel beta sheet, the strands of which are connected by alpha helices. The enzyme differs from other NMP kinases in the presence of a 40-residue insert situated in the NMP-binding (NMPbind) domain. This insert contains two domains: one comprising a three-stranded antiparallel beta sheet, the other comprising two alpha helices. CONCLUSIONS . Two features of the CMP kinase from E. coli have no equivalent in other NMP kinases of known structure. Firstly, the large NMPbind insert undergoes a CDP-induced rearrangement: its beta-sheet domain moves away from the substrate, whereas its helical domain comes closer to it in a motion likely to improve the protection of the active site. Secondly, residues involved in CDP recognition are conserved in CMP kinases and have no counterpart in other NMP kinases. The structures presented here are the first of a new family of NMP kinases specific for CMP.

Simple and fast purification of Escherichia coli adenylate kinase

Adenylate kinase from E. coli (strains CR341 and CR341 T28, a temperature‐sensitive mutant) was purified by a two‐step chromatographic procedure. The enzyme from crude extracts of both mutant and parent strain was bound to blue—Sepharose at pH 7.5, thereafter specifically eluted with 0.05 mM P 1,P 5‐di(adenosine‐5′)pentaphosphate. A second chromatography on Sephadex G‐100 yielded pure enzyme. E. coli adenylate kinase was strongly inhibited by P 1,P 5‐di(adenosine‐5′)pentaphosphate (K i 0.6 μM for adenylate kinase of strain CR341 and 2.1 μM in the case of mutant enzyme). After denaturation in 6 M guanidinium hydrochloride both mutant and parent adenylate kinase returned rapidly to the native, active state by dilution of guanidinium hydrochloride.

Properties of a human cytomegalovirus-induced protein kinase

A human cytomegalovirus (HCMV)-induced polypeptide of 68,000 Da (p68) with protein kinase activity was identified using a monoclonal antibody (F6b) produced against HCMV-infected cell proteins. p68 was detected by immunoprecipitation from 3 to 120 hr after infection and was induced by several strains of human and not simian CMV. Protein kinase activity was associated almost exclusively with nuclear HCMV-induced p68. Enzyme activity with ATP and casein as phosphate donor and acceptor, respectively, exhibited an optimum pH between 6 and 6.5. It was Mg2+ dependent and cAMP independent. The KATPm of 45 microM at pH 6.5 indicated a relatively high affinity of p68 for the nucleotide. p68 also transferred phosphate to phosvitin and light chains of F6b, as well as autophosphorylating at threonine and serine residues.

Structural and nucleotide-binding properties of YajQ and YnaF, two Escherichia coli proteins of unknown function

Structural genomics is a new approach in functional assignment of proteins identified via whole‐genome sequencing programs. Its rationale is that nonhomologous proteins performing similar or related biological functions might have similar tertiary structure. We used dye pseudoaffinity chromatography, two‐dimensional gel electrophoresis, and mass spectrometry to identify two novel Escherichia coli nucleotide‐binding proteins, YnaF and YajQ. YnaF exhibited significant sequence identity with MJ0577, an ATP‐binding protein from a hyperthermophile (Methanococcus jannaschii), and with UspA, a protein from Haemophilus influenzae that belongs to the Universal Stress Protein family. YnaF conserves the ATP‐binding site and the dimeric structure observed in the crystal of MJ0577. The protein YajQ, present in many bacterial genomes, is missing in eukaryotes. In the absence of significant similarities of YajQ to any solved structure, we determined its structural and ligand‐binding properties by NMR and isothermal titration calorimetry. We demonstrate that YajQ is composed of two domains, each centered on a β‐sheet, that are connected by two helical segments. NMR studies, corroborated with local sequence conservation among YajQ homologs in various bacteria, indicate that one of the β‐sheets is mostly involved in biological activity.

Spectrophotometric method for assay of mitochondrial oxygen uptake using oxyhemoglobin as indicator and oxygen donor.

Abstract HbO2 dissociation in an adequate medium and in the presence of respiratory enzymes was investigated by a spectrokinetic method. In a kinetic way it is possible to establish a precise relation between the rate of reduction of HbO2 (measured spectrophotometrically) and the rate of mitochondrial oxygen uptake. At HbO2 concentrations greater than 0.2 meq/liter the rate of HbO2 reduction depends only on mitochondrial concentration and temperature. The authors show why the HbO2 reduction may be utilized to test the mitochondrial (or other biological samples) oxygen uptake as well as the phosphorylation ratio.

Spectrophotometric method for assay of mitochondrial oxygen uptake: II. Simultaneous determination of mitochondrial swelling, respiration, and phosphate esterification

Abstract An improved spectrophotometric method for the assay of mitochondrial oxygen uptake is presented. This is based on oxyhemoglobin utilization as oxygen donor and as indicator. By simultaneous determinations at two wavelengths (560 and 547.5 mμ) we may follow the mitochondrial oxygen uptake and the volume changes due to swelling or contraction processes. Allowing the possibility of some additional assays such as inorganic phosphate esterification, substrate consumption rate, and mitochondrial exchanges of ions, the spectrophotometric method furnishes additional data for comparison with manometry or polarography. This advantage should have influence in going deeply into quantitative studies of the polymodality of energy transformations of the mitochondrial respiratory chain.

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

Calmodulin‐activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13–24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site‐directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin‐binding properties of mutated proteins showed no important differences when compared to the wild‐type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3′‐anthraniloyl‐2′‐deoxyadenosine 5′‐triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP‐utilizing enzymes, are essential for binding the Mg(2+)‐nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide‐binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.

Two-Dimensional Infrared Correlation Analysis of Protein Unfolding: Use of Spectral Simulations to Validate Structural Changes during Thermal Denaturation of Bacterial CMP Kinases

The functional role of bacterial CMP kinases is to recover the energetically exhausted nucleoside monophosphates derived from cell metabolism by transferring a phosphate residue from ATP to CMP or dCMP. These enzymes—important for cell growth and division—possess two distinct binding sites and a number of conserved secondary structure elements. Herein we compare the infrared spectra of two similar, but not identical, CMP kinases from Escherichia coli and Bacillus subtilis. The two-dimensional correlation analysis of the infrared spectra of the two enzymes reveals significant differences in protein structure upon denaturation, a fact possibly linked to their different biochemical and catalytic properties. Model calculations are used to illustrate the effect of two separate processes on the out-of-phase correlation in the two-dimensional (2D) correlation plots. This strategy is then employed to validate the changes observed in the secondary structure of the two enzymes. When bound to the active site of the protein, the two substrates CMP and ATP exert a stabilizing effect on the structure of both proteins; however, the changes observed upon thermal denaturation are different for the two enzymes. Model 2D correlations that simulate the denaturation of the two enzymes confirm the occurrence of temperature-delayed unfolding processes in both proteins. Thermal denaturation and aggregation can be distinguished in both proteins as two distinct processes, separated in time.

Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene renders Yersinia pestis avirulent

Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Yersinia pestis, the causative agent of plague, belongs to this family. However, it was previously shown that its thymidylate kinase (TMPKyp) exhibits biochemical properties significantly different from those of its Escherichia coli counterpart [Chenal-Francisque, Tourneux, Carniel, Christova, Li de la Sierra, Barzu and Gilles (1999) Eur. J. Biochem. 265, 112-119]. In this work, the adenylate kinase (AK) of Y. pestis (AKyp) was characterized. As with TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (Ser129Phe and Pro87Ser), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the Pro87Ser substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37 degree C. In virulence testings, less than 50 colony forming units (CFU) of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, whereas bacterial loads as high as 1.5 x 10(4) CFU of the adk mutant were unable to kill any animals.