Activation of a cyanobacterial adenylate cyclase, CyaC, by autophosphorylation and a subsequent phosphotransfer reaction.
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The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced in Escherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylate in vitro with the gamma-phosphate of [gamma-32P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895. The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced. Asp895 is phosphorylated by acetyl [32P]phosphate, a small phosphoryl molecule, but Asp60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact. These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity. (+info)
Btf, a novel death-promoting transcriptional repressor that interacts with Bcl-2-related proteins.
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The adenovirus E1B 19,000-molecular-weight (19K) protein is a potent inhibitor of apoptosis and cooperates with E1A to transform primary rodent cells. E1B 19K shows sequence and functional homology to the mammalian antiapoptotic gene product, Bcl-2. Like Bcl-2, the biochemical mechanism of E1B 19K function includes binding to and antagonization of cellular proapoptotic proteins such as Bax, Bak, and Nbk/Bik. In addition, there is evidence that E1B 19K can affect gene expression, but whether this contributes to its antiapoptotic function has not been determined. In an effort to further understand the functions of E1B 19K, we screened for 19K-associated proteins by the yeast two-hybrid system. A novel protein, Btf (Bcl-2-associated transcription factor), that interacts with E1B 19K as well as with the antiapoptotic family members Bcl-2 and Bcl-xL but not with the proapoptotic protein Bax was identified. btf is a widely expressed gene that encodes a protein with homology to the basic zipper (bZip) and Myb DNA binding domains. Btf binds DNA in vitro and represses transcription in reporter assays. E1B 19K, Bcl-2, and Bcl-xL sequester Btf in the cytoplasm and block its transcriptional repression activity. Expression of Btf also inhibited transformation by E1A with either E1B 19K or mutant p53, suggesting a role in either promotion of apoptosis or cell cycle arrest. Indeed, the sustained overexpression of Btf in HeLa cells induced apoptosis, which was inhibited by E1B 19K. Furthermore, the chromosomal localization of btf (6q22-23) maps to a region that is deleted in some cancers, consistent with a role for Btf in tumor suppression. Thus, btf may represent a novel tumor suppressor gene residing in a unique pathway by which the Bcl-2 family can regulate apoptosis. (+info)
Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins.
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The terminase enzyme from bacteriophage lambda is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage lambda and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA subunits. We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate. Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step. Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme. The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor. The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies. (+info)
Evidence for a copper-coordinated histidine-tyrosine cross-link in the active site of cytochrome oxidase.
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Following hints from X-ray data (Ostermeier C et al., 1997, Proc Natl Acad Sci USA 94:10547-10553; Yoshikawa S et al., 1998, Science 280: 1723-1729), chemical evidence is presented from four distantly related cytochrome-c oxidases for the existence of a copperB-coordinated His240-Tyr244) cross-link at the O2-activating Heme Fea3-CuB center in the catalytic subunit 1 of the enzyme. The early evolutionary invention of this unusual structure may have prevented damaging *OH-radical release at e(-)-transfer to dioxygen and thus have enabled O2 respiration. (+info)
UDP-N-acetylglucosamine:N-acetylmuramoyl-(pentapeptide) pyrophosphoryl undecaprenol N-acetylglucosamine transferase from Escherichia coli: overproduction, solubilization, and purification.
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Plasmids for the high-level overproduction of wild-type, and C- and N-terminal His-tagged MurG N-acetylglucosaminyl transferase from Escherichia coli were constructed. In complementation tests the three forms were active in vivo. After IPTG induction, growth, spheroplast formation and lysis, overproduced MurG proteins were mainly present (90%) in the particulate fraction. Readily solubilized by CHAPS, they were purified without any detergent to over 80% purity for both His-tagged forms but only up to 20% for the wild-type form. The enzymatic activity of each purified MurG protein was determined and found to be inhibited to the same extent by ramoplanin. (+info)
Zinc coordination and substrate catalysis within the neuropeptide processing enzyme endopeptidase EC 3.4.24.15. Identification of active site histidine and glutamate residues.
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Endopeptidase EC 3.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is broadly distributed within the brain, pituitary, and gonads. Its substrate specificity includes a number of physiologically important neuropeptides such as neurotensin, bradykinin, and gonadotropin-releasing hormone, the principal regulatory peptide for reproduction. In studying the structure and function of EP24.15, we have employed in vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme and allow us to glean insight into the mechanism of substrate binding and catalysis. Comparison of the sequence of EP24.15 with bacterial homologues previously solved by x-ray crystallography and used as models for mammalian metalloendopeptidases, indicates conserved residues. The active site of EP24.15 exhibits an HEXXH motif, a common feature of zinc metalloenzymes. Mutations have confirmed the importance, for binding and catalysis, of the residues (His473, Glu474, and His477) within this motif. A third putative metal ligand, presumed to coordinate directly to the active site zinc ion in concert with His473 and His477, has been identified as Glu502. Conservative alterations to these residues drastically reduces enzymatic activity against both a putative physiological substrate and a synthetic quenched fluorescent substrate as well as binding of the specific active site-directed inhibitor, N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate, the binding of which we have shown to be dependent upon the presence, and possibly coordination, of the active site zinc ion. These studies contribute to a more complete understanding of the catalytic mechanism of EP24.15 and will aid in rational design of inhibitors and pharmacological agents for this class of enzymes. (+info)
Site-directed mutagenesis of putative substrate-binding residues reveals a mechanism controlling the different stereospecificities of two tropinone reductases.
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Two tropinone reductases (TRs) constitute a key branch point in the biosynthetic pathway of tropane alkaloids, which are mainly produced in several solanaceous plants. The two TRs share 64% identical amino acid residues and reduce the 3-carbonyl group of a common substrate, tropinone, but they produce distinct alcohol products with different stereospecific configurations. Previous x-ray crystallographic analysis has revealed their highly conserved overall folding, and the modeling of tropinone within the putative substrate-binding sites has suggested that the different stereospecificities may be determined solely by the different binding orientations of tropinone to the enzymes. In this study, we have constructed various mutant TRs, in which putative substrate-binding residues from one TR were substituted with those found in the corresponding positions of the other TR. Substitution of five amino acid residues resulted in an almost complete reversal of stereospecificity, indicating that the different stereospecificities are indeed determined by the binding orientation of tropinone. Detailed kinetic analysis of the mutant enzymes has shown that TR stereospecificity is determined by varying the contributions from electrostatic and hydrophobic interactions and that the present TR structures represent highly evolved forms, in which strict stereospecificities and rapid turnover are accomplished together. (+info)
A fission yeast gene (prr1(+)) that encodes a response regulator implicated in oxidative stress response.
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An inspection of the Schizosaccharomyces pombe genome database revealed that this eukaryotic microorganism possesses a gene that may encode a bacterial type of histidine-to-aspartate (His-Asp) phosphorelay component, namely, a response regulator. The predicted gene, named prr1(+) (S. pombe response regulator), encodes a protein that contains a typical phospho-accepting receiver domain, preceded by a mammalian heat shock factor (HSF)-like DNA-binding domain. Inactivation of this prr1(+) gene resulted in mutant cells defective in some aspects of stress responses, including sensitivity to oxidative stress, cold-temperature, and heavy metal toxicity. It was also demonstrated that Prr1 is required for the transcription of some genes (e.g., trr1(+), ctt1(+)), which are induced by oxidative stress. These results suggest that a His-Asp phosphorelay system may be involved in a stress-activated signaling pathway in S. pombe. (+info)