Structural and functional characterization of Escherichia coli UMP kinase in complex with its allosteric regulator GTP.

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of UTP. They are hexamers regulated by GTP (allosteric activator) and UTP (inhibitor). We describe here the 2.8 angstroms crystal structure of Escherichia coli UMP kinase bound to GTP. The GTP-binding site, situated at 15 angstroms from the UMP-binding site and at 24 angstroms from the ATP-binding site, is delineated by two contiguous dimers. The overall structure, as compared with those bound to UMP, UDP, or UTP, shows a rearrangement of its quaternary structure: GTP induces an 11 degrees opening of the UMP kinase dimer, resulting in a tighter dimer-dimer interaction. A nucleotide-free UMP kinase dimer has an intermediate opening. Superposition of our structure with that of archaeal UMP kinases, which are also hexamers, shows that a loop appears to hamper any GTP binding in archeal enzymes. This would explain the absence of activating effect of GTP on this group of UMP kinases. Among GTP-binding residues, the Asp-93 is the most conserved in bacterial UMP kinases. In the previously published structures of E. coli UMP kinase, this residue was shown to be involved in hydrogen bonds between the subunits of a dimer. Its substitution by an alanine decreases the cooperativity for UTP binding and suppresses the reversal by GTP of UTP inhibition. This demonstrates that the previously described mutual exclusion of these two nucleotides is mediated by Asp-93.

Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria.

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.

Conversion of methionine to cysteine in Bacillus subtilis and its regulation.

Bacillus subtilis can use methionine as the sole sulfur source, indicating an efficient conversion of methionine to cysteine. To characterize this pathway, the enzymatic activities of CysK, YrhA and YrhB purified in Escherichia coli were tested. Both CysK and YrhA have an O-acetylserine-thiol-lyase activity, but YrhA was 75-fold less active than CysK. An atypical cystathionine beta-synthase activity using O-acetylserine and homocysteine as substrates was observed for YrhA but not for CysK. The YrhB protein had both cystathionine lyase and homocysteine gamma-lyase activities in vitro. Due to their activity, we propose that YrhA and YrhB should be renamed MccA and MccB for methionine-to-cysteine conversion. Mutants inactivated for cysK or yrhB grew similarly to the wild-type strain in the presence of methionine. In contrast, the growth of an DeltayrhA mutant or a luxS mutant, inactivated for the S-ribosyl-homocysteinase step of the S-adenosylmethionine recycling pathway, was strongly reduced with methionine, whereas a DeltayrhA DeltacysK or cysE mutant did not grow at all under the same conditions. The yrhB and yrhA genes form an operon together with yrrT, mtnN, and yrhC. The expression of the yrrT operon was repressed in the presence of sulfate or cysteine. Both purified CysK and CymR, the global repressor of cysteine metabolism, were required to observe the formation of a protein-DNA complex with the yrrT promoter region in gel-shift experiments. The addition of O-acetyl-serine prevented the formation of this protein-DNA complex.

Structure of Escherichia coli UMP kinase differs from that of other nucleoside monophosphate kinases and sheds new light on enzyme regulation.

Bacterial UMP kinases are essential enzymes involved in the multistep synthesis of nucleoside triphosphates. They are hexamers regulated by the allosteric activator GTP and inhibited by UTP. We solved the crystal structure of Escherichia coli UMP kinase bound to the UMP substrate (2.3 A resolution), the UDP product (2.6 A), or UTP (2.45 A). The monomer fold, unrelated to that of other nucleoside monophosphate kinases, belongs to the carbamate kinase-like superfamily. However, the phosphate acceptor binding cleft and subunit assembly are characteristic of UMP kinase. Interactions with UMP explain the high specificity for this natural substrate. UTP, previously described as an allosteric inhibitor, was unexpectedly found in the phosphate acceptor site, suggesting that it acts as a competitive inhibitor. Site-directed mutagenesis of residues Thr-138 and Asn-140, involved in both uracil recognition and active site interaction within the hexamer, decreased the activation by GTP and inhibition by UTP. These experiments suggest a cross-talk mechanism between enzyme subunits involved in cooperative binding at the phosphate acceptor site and in allosteric regulation by GTP. As bacterial UMP kinases have no counterpart in eukaryotes, the information provided here could help the design of new antibiotics.

High-level production of recombinant sulfide-reactive hemoglobin I from Lucina pectinata in Escherichia coli. High yields of fully functional holoprotein synthesis in the BLi5 E. coli strain.

Hemoglobin I (HbI) from Lucina pectinata is a monomeric protein composed of 143 amino acids with high sulfide affinity. Its unique heme pocket contains three residues not commonly found in vertebrate globins: Phe 29 (B10), Gln 64 (E7), and Phe 68 (E11), which are thought to be important for high affinity for hydrogen sulfide. Recombinant HbI (rHbI) and several site-directed mutants were cloned and expressed in Escherichia coli yielding high amounts of protein. The highest rHbI protein yield was obtained when the HbI cDNA was cloned into the pET28 (a+) expression vector, transformed into BLi5 cells, the induction performed with 1 mM IPTG at 30 degrees C and TB medium was supplemented with 30 microg/mL hemin chloride and 1% glucose. The highest yield obtained of HbI was 32 mg/L of culture using Fernbach flasks. UV/Visible spectral analysis showed that rHbI binds heme and ESI-MS shows that its molecular weight corresponds to the expected size. Kinetic studies with H2S confirmed that rHbI and HbI have identical binding properties, where the kON for the clam's Hb is 2.73x10(4)M-1s-1 and for rHbI is 2.43x10(4)M-1s-1.

Structure-function relationships of UMP kinases from pyrH mutants of Gram-negative bacteria.

Bacterial uridine monophosphate (UMP) kinases are essential enzymes encoded by pyrH genes, and conditional-lethal or other pyrH mutants were analysed with respect to structure-function relationships. A set of thermosensitive pyrH mutants from Escherichia coli was generated and studied, along with already described pyrH mutants from Salmonella enterica serovar Typhimurium. It is shown that Arg-11 and Gly-232 are key residues for thermodynamic stability of the enzyme, and that Asp-201 is important for both catalysis and allosteric regulation. A comparison of the amino acid sequence of UMP kinases from several prokaryotes showed that these were conserved residues. Discussion on the enzyme activity level in relation to bacterial viability is also presented.

Deciphering the function of an ORF: Salmonella enterica DeoM protein is a new mutarotase specific for deoxyribose.

We identified in Salmonella enterica serovar Typhi a cluster of four genes encoding a deoxyribokinase (DeoK), a putative permease (DeoP), a repressor (DeoQ), and an open reading frame encoding a 337 amino acid residues protein of unknown function. We show that the latter protein, called DeoM, is a hexamer whose synthesis is increased by a factor over 5 after induction with deoxyribose. The CD spectrum of the purified recombinant protein indicated a dominant contribution of betatype secondary structure and a small content of alpha-helix. Temperature and guanidinium hydrochloride induced denaturation of DeoM indicated that the hexamer dissociation and monomer unfolding are coupled processes. DeoM exhibits 12.5% and 15% sequence identity with galactose mutarotase from Lactococcus lactis and respectively Escherichia coli, which suggested that these three proteins share similar functions. Polarimetric experiments demonstrated that DeoM is a mutarotase with high specificity for deoxyribose. Site-directed mutagenesis of His183 in DeoM, corresponding to a catalytically active residue in GalM, yielded an almost inactive deoxyribose mutarotase. DeoM was crystallized and diffraction data collected for two crystal systems, confirmed its hexameric state. The possible role of the protein and of the entire gene cluster is discussed in connection with the energy metabolism of S. enterica under particular growth conditions.

Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy.

The interaction of the adenylate cyclase catalytic domain (AC) of the Bordetella pertussis major exotoxin with its activator calmodulin (CaM) was studied by time-resolved fluorescence spectroscopy using three fluorescent groups located in different regions of AC: tryptophan residues (W69 and W242), a nucleotide analogue (3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, Ant-dATP) and a cysteine-specific probe (acrylodan). CaM binding elicited large changes in the dynamics of W242, which dominates the fluorescence emission of both AC and AC-CaM, similar to that observed for isolated CaM-binding sequences of different lengths [Bouhss, A., Vincent, M., Munier, H., Gilles, A.M., Takahashi, M., Bârzu, O., Danchin, A. & Gallay, J. (1996) Eur. J. Biochem.237, 619-628]. In contrast, Ant-dATP remains completely immobile and inaccessible to the solvent in both the AC and AC-CaM nucleotide-binding sites. As AC contains no cysteine residue, a single-Cys mutant at position 75 was constructed which allowed labeling of the catalytic domain with acrylodan. Its environment is strongly apolar and rigid, and only slightly affected by CaM. The protein's hydrodynamic properties were also studied by fluorescence anisotropy decay measurements. The average Brownian rotational correlation times of AC differed significantly according to the probe used (19 ns for W242, 25 ns for Ant-dATP, and 35 ns for acrylodan), suggesting an elongated protein shape (axial ratio of approximately 1.9). These values increased greatly with the addition of CaM (39 ns for W242, 60-70 ns for Ant-dATP and 56 ns for acrylodan). This suggests that (a) the orientation of the probes is altered with respect to the protein axes and (b) the protein becomes more elongated with an axial ratio of approximately 2.4. For comparison, the hydrodynamic properties of the anthrax AC exotoxin were computed by a mathematical approach (hydropro), which uses the 3D structure [Drum, C.L., Yan, S.-Z., Bard, J., Shen, Y.-Q., Lu, D., Soelalman, S., Grabarek, Z., Bohm, A. & Tang, W.-J. (2002) Nature (London)415, 396-402]. A change in axial ratio is also observed on CaM binding, but in the reverse direction from that for AC: from 1.7 to 1.3. The mechanisms of activation of the two proteins by CaM may therefore be different.

Regulation of expression of the 2-deoxy-D-ribose utilization regulon, deoQKPX, from Salmonella enterica serovar typhimurium.

Salmonella enterica, in contrast to Escherichia coli K12, can use 2-deoxy-D-ribose as the sole carbon source. The genetic determinants for this capacity in S. enterica serovar Typhimurium include four genes, of which three, deoK, deoP, and deoX, constitute an operon. The fourth, deoQ, is transcribed in the opposite direction. The deoK gene encodes deoxyribokinase. In silico analyses indicated that deoP encodes a permease and deoQ encodes a regulatory protein of the deoR family. The deoX gene product showed no match to known proteins in the databases. Deletion analyses showed that both a functional deoP gene and a functional deoX gene were required for optimal utilization of deoxyribose. Using gene fusion technology, we observed that deoQ and the deoKPX operon were transcribed from divergent promoters located in the 324-bp intercistronic region between deoQ and deoK. The deoKPX promoter was 10-fold stronger than the deoQ promoter, and expression was negatively regulated by DeoQ as well as by DeoR, the repressor of the deoxynucleoside catabolism operon. Transcription of deoKPX but not of deoQ was regulated by catabolite repression. Primer extension analysis identified the transcriptional start points of both promoters and showed that induction by deoxyribose occurred at the level of transcription initiation. Gel retardation experiments with purified DeoQ illustrated that it binds independently to tandem operator sites within the deoQ and deoK promoter regions with K(d) values of 54 and 2.4 nM, respectively.

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.

UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity.

The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly. Binding of Ant-dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.

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 beta-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 beta-sheets is mostly involved in biological activity.

Comparative modelling and immunochemical reactivity of Escherichia coli UMP kinase.

Bacterial UMP kinases do not exhibit any sequence homology with other nucleoside monophosphate kinases described so far, and appear under oligomeric forms, submitted to complex regulation by nucleotides. We propose here a structural model of UMP kinase from Escherichia coli based on the conservation of the fold of carbamate kinase whose crystal structure was recently solved. Despite sequence identity of only 18% over 203 amino acids, alignment of UMP kinase from E. coli with carbamate kinase from Enterococcus faecalis by hydrophobic cluster analysis and threading suggested the conservation of the overall structure, except for a small subdomain (absent in UMP kinase). The modelled dimer suggested conservation of the dimer interface observed in carbamate kinase while interaction of UMP kinase with a monoclonal antibody (Mab 44-2) suggests a three in-plane dimer subunit arrangement. The model was analyzed in light of various modified forms of UMP kinase obtained by site-directed mutagenesis.

Sugar specificity of bacterial CMP kinases as revealed by crystal structures and mutagenesis of Escherichia coli enzyme.

Bacterial cytidine monophosphate (CMP) kinases are characterised by an insert enlarging their CMP binding domain, and by their particular substrate specificity. Thus, both CMP and 2'-deoxy-CMP (dCMP) are good phosphate acceptors for the CMP kinase from Escherichia coli (E. coli CMPK), whereas eukaryotic UMP/CMP kinases phosphorylate the deoxynucleotides with very low efficiency. Four crystal structures of E. coli CMPK complexed with nucleoside monophosphates differing in their sugar moiety were solved. Both structures with CMP or dCMP show interactions with the pentose that were not described so far. These interactions are lost with the poorer substrates AraCMP and 2',3'-dideoxy-CMP. Comparison of all four structures shows that the pentose hydroxyls are involved in ligand-induced movements of enzyme domains. It also gives a structural basis of the mechanism by which either ribose or deoxyribose can be accommodated. In parallel, for the four nucleotides the kinetic results of the wild-type enzyme and of three structure-based variants are presented. The phosphorylation rate is significantly decreased when either of the two pentose interacting residues is mutated. One of these is an arginine that is highly conserved in all known nucleoside monophosphate kinases. In contrast, the other residue, Asp185, is typical of bacterial CMP kinases. It interacts with Ser101, the only residue conserved in all CMP binding domain inserts. Mutating Ser101 reduces CMP phosphorylation only moderately, but dramatically reduces dCMP phosphorylation. This is the first experimental evidence of a catalytic role involving the characteristic insert of bacterial CMP kinases. Furthermore, this role concerns only dCMP phosphorylation, a feature of this family of enzymes.