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.

Dictyostelium nucleoside diphosphate kinase highly homologous to Nm23 and Awd proteins involved in mammalian tumor metastasis and Drosophila development.

Two complementary DNAs (cDNAs) previously isolated, one by functional screening and the other by immunological screening of a Dictyostelium discoideum expression library, encode two proteins, Gip17 and Guk7.2, sharing 71% homology. In the present study, we found that the expression of their messenger RNAs (mRNAs) is developmentally regulated, with a sharp decrease during the first hours of differentiation. The Gip17 protein was purified to homogeneity from D. discoideum amoebas and from recombinant Escherichia coli and was conclusively identified as a nucleoside diphosphate (NDP) kinase. NDP kinases play a major role in synthesis of nucleoside triphosphates and, in many systems, are found associated with guanosine triphosphate (GTP)-binding proteins. We found the Gip17 protein to be 77% homologous to the human Nm23 protein and 75% homologous to the Drosophila melanogaster Awd protein. The levels of murine and human nm23 mRNA and Nm23 protein are significantly reduced in tumor cells of high metastatic potential, suggesting that Nm23 is involved in suppression of mammalian tumor metastasis, and mutants of the awd gene exhibit widespread development abnormalities, suggesting that Awd is involved in D. melanogaster development. The high percentage of homology of the Gip17 and Guk7.2 proteins with the Nm23 and Awd proteins indicates that Nm23 and Awd also have nucleoside diphosphate kinase activity. Possible modulations in the activity of this metabolic enzyme could be related to the altered metabolism of tumor cells and the control of metastatic potential. Our results point to an unexpected role of NDP kinase in development, growth control, and oncogenic transformation.

Pressure-induced changes in the secondary structure of the Escherichia coli methionine repressor protein.

The effect of hydrostatic pressure on the conformational properties of the E. coli methionine repressor protein in aqueous solution was investigated by infrared spectroscopy. Changes in hydrostatic pressure produce dramatic changes in the spectral region of the conformation-sensitive amide I band. As the pressure is raised up to 18 kbar, the protein undergoes a rearrangement of alpha-helical segments into beta-type structures; after the pressure is released the beta-strands reconvert into less ordered alpha-helical or random segments.

Regulatory effects of purine nucleotide analogs with liver glutamate dehydrogenase.

A total of 26 different purine nucleotides with specific modifications in the base moiety and/or in the polyphosphate chain as well as various combinations of nucleotides were tested as allosteric effectors of beef liver glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3). The capacity of these nucleotide analogs to activate or to inhibit the glutamate dehydrogenase activity is expressed quantitatively and scaled between the extreme effects of ADP and GTP, respectively. The significance of distinct structural elements for the enzyme-effector interaction is discussed. While the inhibitory GTP site is less specific, accepting many natural and most modified nucleoside triphosphates as inhibitors, the activating ADP site shows a much higher specificity for nucleotides as activators.

Catalytic properties of Sepharose-bound L-alanine dehydrogenase from Bacillus cereus.

(1) L-Alanine dehydrogenase from Bacillus cereus was purified by a two-step chromatographic procedure involving Cibacron-Blue 3G-A Sepharose 4B-CL, and Sepharose 6B-CL, and immobilized on CNBr-activated Sepharose 4B. (2) Following immobilization via two of the six subunits, L-alanine dehydrogenase retained 66% of the specific activity of the soluble enzyme. The affinity of the immobilized enzyme for NH4+, pyruvate and L-alanine, was not different to that of the soluble form. The Km of the Sepharose-bound L-alanine dehydrogenase for pyridine coenzymes was 6-8-times higher than in the soluble case. (3) The stability of L-alanine dehydrogenase towards urea or thermal denaturation was increased by immobilization. (4) The incubation at 37 degrees C for 24 h of the immobilized L-alanine dehydrogenase with 3 M NH4Cl/NH4OH buffer (pH 9) released 70% of the enzyme. The specific activity and the affinity of the 'solubilized' L-alanine dehydrogenase for the pyridine coenzymes was the same as that obtained with the original, soluble L-alanine dehydrogenase.

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation.

Various analogs of adenosine 5'-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles. It is shown that the fluorophosphate analog ATP(gamma F) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(gamma F) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions. The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(gamma F), it is strong inhibitor of both soluble and membrane-bound mitochondrial ATPases. The biological implication of the complementary effects of ATP(gamma F) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.

Nucleotide specificity of pyruvate kinase and phosphoenolpyruvate carboxykinase.

Various analogues of adenosine 5'-diphosphate with modifications in the heterocyclic base residue were tested as substrates of rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC. 2.7.1.40) and guinea pig liver mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32). The significance of different structural elements for the enzyme-substrate interaction is discussed. While pyruvate kinase shows a rather broad specificity for these analogues, phosphoenolpyruvate carboxykinase has a more stringent requirement for nucleotides, the intact keto and NH groups at C6 and N1 of the pyrimidine ring representing essential sites for the phosphoenolpyruvate carboxykinase substrate interaction. The biological significance of the different substrate specificities of pyruvate kinase and phosphoenolpyruvate carboxykinase is discussed as a possible metabolic control factor.

X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 A resolution.

The X-ray structure of Mycobacterium tuberculosis TMP kinase at 1.95 A resolution is described as a binary complex with its natural substrate TMP. Its main features involve: (i) a clear magnesium-binding site; (ii) an alpha-helical conformation for the so-called LID region; and (iii) a high density of positive charges in the active site. There is a network of interactions involving highly conserved side-chains of the protein, the magnesium ion, a sulphate ion mimicking the beta phosphate group of ATP and the TMP molecule itself. All these interactions conspire in stabilizing what appears to be the closed form of the enzyme. A complete multialignment of all (32) known sequences of TMP kinases is presented. Subtle differences in the TMP binding site were noted, as compared to the Escherichia coli, yeast and human enzyme structures, which have been reported recently. These differences could be used to design specific inhibitors of this essential enzyme of nucleotide metabolism. Two cases of compensatory mutations were detected in the TMP binding site of eukaryotic and prokaryotic enzymes. In addition, an intriguing high value of the electric field is reported in the vicinity of the phosphate group of TMP and the putative binding site of the gamma phosphate group of ATP.

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 P1,P5-di(adenosine-5')pentaphosphate. A second chromatography on Sephadex G-100 yielded pure enzyme. E. coli adenylate kinase was strongly inhibited by P1,P5-di(adenosine-5')pentaphosphate (Ki 0.6 microM for adenylate kinase of strain CR341 and 2.1 microM 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.

Asparagine-135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket.

This work studies the structure-function relationships of Asn135, a residue situated in the GTP binding pocket of elongation factor Tu (EF-Tu). For this purpose we constructed EF-TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF-TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn135 side-chain does not recognize the exocyclic keto group of the guanine base. However, EF-TuN135D/D138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa-tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn135 is important for the correct folding of the nucleotide binding site and that EF-Tu.kirromycin can mediate the binding of aa-tRNA to the mRNA-programmed ribosomes independently of the native conformation of this site.

Borrelia burgdorferi uridine kinase: an enzyme of the pyrimidine salvage pathway for endogenous use of nucleotides.

The 621 bp udk gene encoding Borrelia burgdorferi potential uridine kinase, involved in the pyrimidine salvage pathway, was cloned and sequenced. The B burgdorferi protein has a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel. The N-terminal sequence of the protein, Ala-Lys-Ile-Ile, is identical to that predicted but lacks N-terminal methionine. udk is located at around 15 kb from the left telomere and forms an operon with an upstream ORF. A likely hypothesis for the role of the pyrimidine salvage pathway is the sole use of endogenous nucleotides for Borrelia.

Bordetella pertussis adenylate cyclase: the gene and the protein.

Using the adenylate cyclase-calmodulin interaction as a tool, the B. pertussis cya gene was cloned in a cya defective E. coli strain harbouring a plasmid which expressed high levels of calmodulin. The determination of the nucleotide sequence of the gene showed that adenylate cyclase is synthesized as a large precursor of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 400 amino acids whereas the 1300 amino acid carboxy-terminal part of the precursor is endowed with haemolytic activity. The catalytically active 43 kDa form of adenylate cyclase is organized in two domains: the N-terminal domain of 25 kDa harbors the catalytic site, and the 18 kDa C-terminal domain carries the main calmodulin-binding site. Immunological relatedness established between B. pertussis, B. anthracis and rat brain adenylate cyclases suggests a common evolutionary origin of a central domain of these calmodulin-stimulated enzymes. The secretion of the adenylate cyclase-haemolysin bifunctional protein (cyclolysin) requires the expression of three additional genes, contiguous to the cya gene. These four genes appear to form a single operon. The mechanism of secretion of the bifunctional protein should be similar to that described for E. coli alpha-haemolysin.