Methionine regeneration and aminotransferases in Bacillus subtilis,Bacillus cereus,and Bacillus anthracis

The conversion of ketomethiobutyrate to methionine has been previously examined in a number of organisms, wherein the aminotransferases responsible for the reaction have been found to be members of the Ia subfamily (L. C. Berger, J. Wilson, P. Wood, and B. J. Berger, J. Bacteriol. 183:4421-4434, 2001). The genome of Bacillus subtilis has been found to contain no subfamily Ia aminotransferase sequences. Instead, the analogous enzymes in B. subtilis were found to be members of the If subfamily. These putative aspartate aminotransferases, the yugH, ywfG, ykrV, aspB, and patA gene products, have been cloned, expressed, and characterized for methionine regeneration activity. Only YkrV was able to convert ketomethiobutyrate to methionine, and it catalyzed the reaction only when glutamine was used as amino donor. In contrast, subcellular homogenates of B. subtilis and Bacillus cereus utilized leucine, isoleucine, valine, alanine, phenylalanine, and tyrosine as effective amino donors. The two putative branched-chain aminotransferase genes in B. subtilis, ybgE and ywaA, were also cloned, expressed, and characterized. Both gene products effectively transaminated branched-chain amino acids and ketoglutarate, but only YbgE converted ketomethiobutyrate to methionine. The amino donor preference for methionine regeneration by YbgE was found to be leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. subtilis ybgE gene is a member of the family III of aminotransferases and falls in a subfamily designated here IIIa. Examination of B. cereus and Bacillus anthracis genome data found that there were no subfamily IIIa homologues in these organisms. In both B. cereus and B. anthracis, two putative branched-chain aminotransferases and two putative D-amino acid aminotransferases were discovered as members of subfamily IIIb. These four sequences were cloned from B. cereus, expressed, and characterized. Only the gene product from the sequence designated Bc-BCAT2 was found to convert ketomethiobutyrate to methionine, with an amino donor preference of leucine, isoleucine, valine, phenylalanine, and tyrosine. The B. anthracis homologue of Bc-BCAT2 was also cloned, expressed, and characterized and was found to be identical in activity. The aminooxy compound canaline was found to be an uncompetitive inhibitor of B. subtilis YbgE and also inhibited growth of B. subtilis and B. cereus in culture.     

The heme environment of recombinant human indoleamine 2,3-dioxygenase. Structural properties and substrate-ligand interactions

Indoleamine 2,3-dioxygenase is a heme enzyme that catalyzes the oxidative degradation of L-Trp and other indoleamines. We have used resonance Raman spectroscopy to characterize the heme environment of purified recombinant human indoleamine 2,3-dioxygenase (hIDO). In the absence of L-Trp, the spectrum of the Fe(3+) form displayed six-coordinate, mixed high and low spin character. Addition of L-Trp triggered a transition to predominantly low spin with two Fe-OH(-) stretching modes identified at 546 and 496 cm(-1), suggesting H-bonding between the NH group of the pyrrole ring of L-Trp and heme-bound OH(-). The distal pocket of Fe(3+) hIDO was explored further by an exogenous heme ligand, CN(-); again, binding of L-Trp introduced strong H-bonding and/or steric interactions to the heme-bound CN(-). On the other hand, the spectrum of Fe(2+) hIDO revealed a five-coordinate and high spin heme with or without L-Trp bound. The proximal Fe-His stretching mode, identified at 236 cm(-1), did not shift upon L-Trp addition, indicating that the proximal Fe-His bond strength is not affected by binding of the substrate. The high Fe-His stretching frequency suggests that Fe(2+) hIDO has a strong "peroxidase-like" Fe-His bond. Using CO as a structural probe for the distal environment of Fe(2+) hIDO revealed that binding of L-Trp in the distal pocket converted IDO to a peroxidase-like enzyme. Binding of L-Trp also caused conformational changes to the heme vinyl groups, which were independent of changes of the spin and coordination state of the heme iron. Together these data indicate that the strong proximal Fe-His bond and the strong H-bonding and/or steric interactions between l-Trp and dioxygen in the distal pocket are likely crucial for the enzymatic activity of hIDO.     

Identification of a histamine H4 receptor on human eosinophils--role in eosinophil chemotaxis

Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Galpha15 cells, demonstrates specific binding to histamine with a Kd of 3.28 +/- 0.76 nM and possesses a unique rank order of potency against known histaminergic compounds in a competitive ligand-binding assay (histamine > clobenpropit > iodophenpropit > thioperamide > R-alpha-methylhistamine > cimetidine > pyrilamine). We have therefore termed this receptor human histamine H4. Chemotaxis studies on isolated human eosinophils have confirmed that histamine is chemotactic and that agonists of the known histamine receptors (H1, H2, and H3) do not induce such a response. Furthermore, studies employing histamine-receptor antagonists have shown an inhibition of chemotaxis only by the H3 antagonists clobenpropit and thioperamide. Since these compounds are also antagonists of hH4 we postulate that the receptor mediating histaminergic chemotaxis is this novel histamine H4 receptor.     

Structure of the Golgi and distribution of reporter molecules at 20 degrees C reveals the complexity of the exit compartments

Incubating cells at 20 degrees C blocks transport out of the Golgi complex and amplifies the exit compartments. We have used the 20 degrees C block, followed by EM tomography and serial section reconstruction, to study the structure of Golgi exit sites in NRK cells. The dominant feature of Golgi structure in temperature-blocked cells is the presence of large bulging domains on the three trans-most cisternae. These domains extend laterally from the stack and are continuous with "cisternal" domains that maintain normal thickness and alignment with the other stacked Golgi cisternae. The bulging domains do not resemble the perpendicularly extending tubules associated with the trans-cisternae of control cells. Such tubules are completely absent in temperature-blocked cells. The three cisternae with bulging domains can be identified as trans by their association with specialized ER and the presence of clathrin-coated buds on the trans-most cisterna only. Immunogold labeling and immunoblots show a significant degradation of a medial- and a trans-Golgi marker with no evidence for their redistribution within the Golgi or to other organelles. These data suggest that exit from the Golgi occurs directly from three trans-cisternae and that specialized ER plays a significant role in trans-Golgi function.     

Role of the 14-3-3 C-terminal loop in ligand interaction

14-3-3 proteins are a family of conserved dimeric molecules that interact with a broad range of target proteins, most of which contain phosphoserine/threonine. The amphipathic groove of 14-3-3 is the main structural feature involved in mediating its associations. We have studied another domain of 14-3-3, the C-terminal loop, to determine what role it plays in ligand interaction. A truncated form of 14-3-3zeta lacking this C-terminal loop was generated and found to bind with higher affinity than the wild-type 14-3-3zeta protein to the ligands Raf-1 and Bad. Interestingly, the truncated 14-3-3zeta also showed increased association with the 14-3-3 binding-deficient Bad/S136A mutant. Taken together, these data support a role for the C-terminal loop as a general inhibitor of 14-3-3/ligand interactions. This may provide a mechanism by which inappropriate associations with 14-3-3 are prevented.     

Conformational and molecular modeling studies of sulfated cholecystokinin-15

Conformational features of the C-terminal carboxyamidated pentadecapeptide of CCK (S(19)HRISDRD[SO(4)]-YMGWMDF(33)-NH(2)) were determined by NMR spectroscopy in a zwitterionic membrane-mimetic solvent system, composed of DPC micelles. The C-terminal octapeptide consisted of a well-defined pseudohelix that was nearly identical to the structure previously reported for nonsulfated CCK-8 in the same solvent system. N-terminal amino acids of CCK-15 were highly disordered, with no clear conformational preference. Extensive NOE-restrained molecular dynamics simulations of the CCK-15/CCK(1)-R complex suggested that almost all the experimentally determined intermolecular contact points provided by NMR, site-directed mutagenesis, and photoaffinity labeling could be simultaneously satisfied, when the N-terminus of the ligand is placed in close spatial proximity to the N-terminus of the receptor.     

Kinetic and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).