Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum

A lacto-N-biose phosphorylase (LNBP) was purified from the cell extract of Bifidobacterium bifidum. Its N-terminal and internal amino acid sequences were homologous with those of the hypothetical protein of Bifidobacterium longum NCC2705 encoded by the BL1641 gene. The homologous gene of the type strain B. longum JCM1217, lnpA, was expressed in Escherichia coli to confirm that it encoded LNBP. No significant identity was found with any proteins with known function, indicating that LNBP should be classified in a new family. The lnpA gene is located in a novel putative operon for galactose metabolism that does not contain a galactokinase gene. The operon seems to be involved in intestinal colonization by bifidobacteria mediated by metabolism of mucin sugars. In addition, it may also resolve the question of the nature of the bifidus factor in human milk as the lacto-N-biose structure found in milk oligosaccharides.     

Switching kinetic mechanism and putative proton donor by directed mutagenesis of glutathione reductase

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase, Tyr-177 (the residue corresponding to Tyr-197 in the NADPH-binding pocket of the homologous human enzyme) was changed to phenylalanine (Y177F), serine (Y177S), and glycine (Y177G). The catalytic activity of the Y177F mutant was very similar to that of the wild-type enzyme, but that of the Y177S and Y177G mutants was substantially diminished. However, all three mutants retained the ability to protect the reduced flavin from adventitious oxidation, indicating that Tyr-177 does not act as a simple "lid" on the NADPH-binding pocket and that the protection of the reduced enzyme must be due largely to burial of the isoalloxazine ring in the protein. The wild-type enzyme and Y177F mutant displayed ping-pong kinetics, but the Y177S and Y177G mutants appeared to have switched to an ordered sequential mechanism. This could be explained by supposing that the enzyme normally functions by a hybrid kinetic mechanism and that the Y177S and Y177G mutations diverted flux from the ping-pong loop favored by the wild-type enzyme to an ordered sequential loop. The necessary change in the partitioning of the common E-NADPH intermediate could be caused by a slowing of the formation of the EH2 intermediate on the ping-pong loop, or by the observed concomitant fall in the Km for glutathione favoring flux through the ordered sequential loop. In another experiment, His-439, thought to act as a proton donor/acceptor in the glutathione-binding pocket, was mutated to a glutamine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of proton-pumping rhodopsin lacking a cytoplasmic proton donor residue by X-ray crystallography

DTG/DTS rhodopsin, which was named based on a three-residue motif (DTG or DTS) that is important for its function, is a light-driven proton-pumping microbial rhodopsin using a retinal chromophore. In contrast to other light-driven ion-pumping rhodopsins, DTG/DTS rhodopsin does not have a cytoplasmic proton donor residue, such as Asp, Glu, or Lys. Because of the lack of cytoplasmic proton donor residue, proton directly binds to the retinal chromophore from the cytoplasmic solvent. However, mutational experiments that showed the complicated effects of mutations were not able to clarify the roles played by each residue, and the detail of proton uptake pathway is unclear because of the lack of structural information. To understand the proton transport mechanism of DTG/DTS rhodopsin, here we report the three-dimensional structure of one of the DTG/DTS rhodopsins, PspR from Pseudomonas putida, by X-ray crystallography. We show that the structure of the cytoplasmic side of the protein is significantly different from that of bacteriorhodopsin, the best-characterized proton-pumping rhodopsin, and large cytoplasmic cavities were observed. We propose that these hydrophilic cytoplasmic cavities enable direct proton uptake from the cytoplasmic solvent without the need for a specialized cytoplasmic donor residue. The introduction of carboxylic residues homologous to the cytoplasmic donors in other proton-pumping rhodopsins resulted in higher pumping activity with less pH dependence, suggesting that DTG/DTS rhodopsins are advantageous for producing energy and avoiding intracellular alkalization in soil and plant-associated bacteria.     

Chemical modification of phosphorylase b by tetranitromethane. Identification of a functional tyrosyl residue

Tetranitromethane, C(NO2)4, a reagent for tyrosyl residues, was found to inactivate irreversibly rabbit skeletal muscle glycogen phosphorylase b. Under the chosen conditions seven tyrosyl residues, namely Tyr-75, 203, 262, 280, 403, 552 and 647, were found to be nitrated. Inactivation was prevented by the presence of the allosteric activator 5'-AMP during nitration. Under these latter conditions one of the reactive tyrosyl residues was not modified by C(NO2)4; thus, this residue appeared to be essential for either catalytic activity or allosteric activation. Tryptic digests of phosphorylase b, reacted with C(NO2)4 in the absence and presence of 5'AMP, were fractionated by gel filtration. The peptide mixtures were further purified by reverse-phase HPLC. One of the peptides contained the tyrosyl residue which was modified by C(NO2)4 only in the absence of 5'AMP. The sequence of this peptide was determined. The amino acid residue which is responsible for the loss of activity upon reaction with C(NO2)4 was identified in the amino acid sequence of phosphorylase b as tyrosine-75. Of the other residues modified in the presence and in the absence of C(NO2)4, tyrosine-403 contributes to the glycogen-storage site whereas Tyr-280 is close to the alpha-D-glucose-binding site. These residues, exposed to the solvent both in the presence and in the absence of 5'AMP, are not essential for catalytic activity.     

Identification of a putative collagen-binding protein from chicken skeletal muscle as glycogen phosphorylase.

We have purified and generated antisera to a 95 kDa skeletal muscle protein that constitutes the largest mass fraction of gelatin-agarose binding proteins in skeletal muscle. Preliminary results indicated that this 95 kDa chicken skeletal muscle protein bound strongly to gelatin-agarose and type IV collagen-agarose, suggesting a possible function in muscle cell adhesion to collagen. However, N-terminal sequencing of proteolytic fragments of the 95 kDa protein indicates that it is the chicken skeletal muscle form of glycogen phosphorylase, the binding of which to gelatin-agarose is unlikely to be biologically relevant. Further characterization showed that the skeletal muscle form of glycogen phosphorylase is immunologically distinct from the liver and brain forms in the chicken, and suggests that, unlike mammalian skeletal muscle, chicken skeletal muscle may have two phosphorylase isoforms. Furthermore, immunolocalization data and solubility characteristics of glycogen phosphorylase in muscle extraction experiments suggest the enzyme may interact strongly with an unidentified component of the muscle cytoskeleton. Thus, this study yields a novel purification technique for skeletal muscle glycogen phosphorylase, provides new information on the distribution and isoforms of glycogen phosphorylase, and provides a caveat for using gelatin affinity chromatography as a primary step in purifying collagen-binding proteins from skeletal muscle.     

Identification of the pH-dependent membrane anchor of carboxypeptidase E (EC 3.4.17.10)

Carboxypeptidase E (CPE), a peptide hormone-processing enzyme, is present within secretory granules in both a soluble form and a form which is membrane-bound at pH 5.5 but soluble at neutral pH. Antisera raised against a peptide corresponding to the predicted COOH-terminus of CPE bind to the membrane-associated form of CPE but not to the soluble form. This COOH-terminal region is predicted to form an amphiphilic alpha-helix, containing several pairs of hydrophobic residues separated by hydrophilic residues. Synthetic COOH-terminal peptides 11-24 residues in length are able to bind to bovine pituitary membranes and can be extracted by conditions that extract the membrane-bound form of CPE. The influence of pH on the membrane binding of a 21-residue COOH-terminal peptide is similar to the membrane binding of CPE: at pH values less than 6 the majority of the peptide is membrane-bound, while at pH values above 8 less than 20% is membrane-bound. Both the 21-residue COOH-terminal peptide and the purified membrane form of CPE, but not the soluble form, partition into Triton X-114 only at low pH (pH less than 6). Combined polar and hydrophobic interactions of the COOH-terminal peptide appear to be responsible for the reversible, pH-dependent association of CPE with membranes.     

Both the dimerization and immunochemical properties of E-cadherin EC1 domain depend on Trp(156) residue

Using site-directed mutagenesis, we show in this paper that the adhesive interface detected in cadherin crystals is unlikely to mediate adhesive interaction between myc- and flag-tagged E-cadherin molecules in human A-431 cells. We also found that a critical residue within this interface, His(233), is part of the epitope for mAb SHE78-7. This epitope was accessible to the antibody in the adhesive E-cadherin dimers, which is consistent with uninvolvement of the site containing His(233) in cell-cell adhesion. However, both the adhesive dimerization and the integrity of the SHE78-7 epitope depended on the same intramolecular interaction between Trp(156) and its hydrophobic pocket. Our data suggest that this interaction may have an important regulatory function in controlling the surface topology of the NH(2)-terminal domain of E-cadherin.     

Lysine 480 is an essential residue in the putative ATP site of lamb kidney (Na,K)-ATPase. Identification of the pyridoxal 5'-diphospho-5'-adenosine and pyridoxal phosphate reactive residue

Pyridoxal 5'-diphospho-5'-adenosine (AP2PL) inhibits lamb kidney (Na,K)-ATPase and that inhibition and covalent modification is blocked by the presence of ATP. After trypsin digestion of the labeled, purified alpha subunit and subsequent peptide mapping of the fluorescently labeled peptides by means of high performance liquid chromatography, the main labeled peptide was further purified and analyzed by amino acid composition analysis and peptide sequencing. The obtained peptide had the sequence Ile470-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-Lys480-Tyr-Gln-Le u-Ser-Ile-His- Lys487. Lysine 480 is the residue modified by AP2PL in the absence, but not in the presence of ATP. The beta subunit is not differentially labeled by AP2PL in the presence or absence of ATP. Interestingly, the same results were obtained using pyridoxal phosphate as the labeling and inactivation reagent, indicating that the specificity of labeling by these reagents is not due to the presence of the adenosine moiety, but instead that the initial recognition of nucleotides by the ATP-binding site of (Na,K)-ATPase may be due to recognition of the phosphate moiety. The amino acid sequence surrounding this lysine residue labeled by both reagents is highly conserved in (Na,K)-ATPase and the related (H,K)-ATPase sequences thus far obtained, which may signify a functional importance for this region of the putative ATP-binding site in these transport proteins.     

Cellobiose phosphorylase (EC 2.4.1.20) of Cellulomonas: occurrence,induction, and its role in cellobiose metabolism

The occurrence of cellobiose cleavage by phosphorolysis and by hydrolysis was investigated in Cellulomonas spec., C. uda, C. flavigena, and C. cartalyticum. Cellobiose phosphorylase (EC 2.4.1.20) was shown to be produced by Cellulomonas spec. when cellobiose or cellulose was used as sole source of energy and carbon but not with glycerol or glucose. Using inhibitors of protein synthesis as well as double labelling techniques it was shown that cellobiose phosphorylase is synthesized de novo in Cellulomonas spec. Aryl--D-glucosidase which was shown to be present in crude extracts of this microorganism as well is not involved in cellobiose cleavage.Abbreviations oNPGluc ortho-nitrophenyl--D-glucopyranoside - oNPGlucase ortho-nitrophenyl--D-glucopyranoside hydrolase (aryl--D-glucosidase) - CMC carboxymethyl-cellulose - CMCase carboxymethyl-cellulase - PAGE polyacrylamde disc gel electrophoresis Parts of this work were presented on the Herbsttagung der Gesellschaft für Biologische Chemie (Schimz et al. 1979) and on the 14th FEBS Meeting (Schimz et al. 1981)     

The Streptomyces coelicolor polynucleotide phosphorylase homologue, and not the putative poly(A) polymerase, can polyadenylate RNA

A protein containing a nucleotidyltransferase motif characteristic of poly(A) polymerases has been proposed to polyadenylate RNA in Streptomyces coelicolor (P. Bralley and G. H. Jones, Mol. Microbiol. 40:1155-1164, 2001). We show that this protein lacks poly(A) polymerase activity and is instead a tRNA nucleotidyltransferase that repairs CCA ends of tRNAs. In contrast, a Streptomyces coelicolor polynucleotide phosphorylase homologue that exhibits polyadenylation activity may account for the poly(A) tails found in this organism.     

Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe.

cDNA encoding Schizosaccharomyces pombe alpha-glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino-acid residues with M(r) 106 138. The deduced amino-acid sequence showed a high homology to those of alpha-glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the alpha-glucosidase family II. By site-directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the alpha-glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (-COO-) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.     

Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum

We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-phosphate; the lnpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine 1-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.     

Identification of cytosolic leucyl aminopeptidase (EC 3.4.11.1) as the major cysteinylglycine-hydrolysing activity in rat liver

Cysteinylglycine hydrolysis is a step in the metabolism of glutathione and glutathione S-conjugates. We had previously observed that in rat liver the enzymatic activity is predominantly located in the cytosol. Here we demonstrate that cytosolic leucyl aminopeptidase (EC 3.4.11.1) is the major cysteinylglycine hydrolysing activity in rat liver. Evidence was obtained from the use of peptidase inhibitors and from immunoprecipitation studies using Pansorbin-coupled antibodies raised against hog kidney cytosolic leucyl aminopeptidase. Both isolated cytosolic leucyl aminopeptidase and the cysteinylglycine-hydrolysing activity in rat liver cytosol are bound with equal efficiency to the affinity matrix. We demonstrate that cytosolic leucyl aminopeptidase exhibits leucinamidase and cysteinylglycinase activity. Cysteinylglycine, cystinyl-bis-glycine, S-nitrosocysteinylglycine, and bimane-S-cysteinylglycine are hydrolysed at high rates; low activity is seen with leukotriene D4. Our findings establish a previously unrecognised physiological function of cytosolic leucyl aminopeptidase, participating in glutathione metabolism and in the degradation of glutathione S-conjugates via the mercapturic acid pathway.     

Proline residue 280 in the second extracellular loop (EC2) of the VPAC2 receptor is essential for the receptor structure

Inspection of the amino acid sequence of the human VPAC1 and the VPAC2 receptors after alignment of the conserved residues indicates that the second extracellular loop (EC2) is one amino acid shorter in the VPAC1 receptor due to the lack of a proline residue in position 294. We hypothesized that this could be of importance for receptor structure and/or for ligand recognition. Insertion by directed mutagenesis of a proline in that position (294 VPAC1) had little consequence on the binding of several agonists but reduced the affinity for the VPAC1 antagonist. Coupling of the 294 VPAC1 receptor to adenylate cyclase was improved, as demonstrated by an increased affinity for VIP and other agonists, and by a shift of the VPAC1 antagonist to partial agonist behavior. Deletion of the proline 280 (DeltaPro280 VPAC2) in the VPAC2 receptor markedly reduced the apparent affinity for all the agonists tested. Replacement of the proline by a glycine residue had a smaller effect on the ligands affinities. The proline residue in the VPAC2 receptor EC2 is thus essential for the receptor structure, and the EC2 domain is involved in ligand recognition and receptor functionality.     

Identification of the putative mannose 6-phosphate receptor (MPR 46) protein in the invertebrate mollusc

Mannose 6-phosphate receptor (MPR 300) protein was earlier affinity purified on phosphomannan gel from the membrane extracts of whole animal acetone powder of a mollusc, unio, in the presence of EDTA (Udaya Lakshmi, Y., Radha, Y., Hille-Rehfeld, A., von Figura, K., and Siva Kumar, N. (1999) Biosci. Rep. 19:403–409). In the present study we demonstrate that the unio also contains the putative mannose 6-phosphate receptor (MPR 46) that can be purified on the same gel in presence of divalent metal ions (10 mM each of calcium, manganese, and magnesium), and in the absence of sodium chloride and at pH 6.5. Chicken and Fish cell MPR 46 proteins were purified under these conditions (Siva Kumar, N., Udaya Lakshmi, Y., Hille-Rehfeld, A., and von Figura, K. (1999) Comp. Biochem. & Physiol. 123B:261–265). The authenticity of the receptor is further confirmed by its ability to react with the MSC1 antibody that is specific for MPR 46 protein. Additional evidence for the presence of MPR 46 in molluscs could be obtained by metabolic labeling of mollusc cells Biomphalaria glabrata (Bg cells) with [³⁵S] methionine and cysteine, and passing the labeled membrane extract on phosphomannan gel (at pH 6.5 and 7.0). On elution with mannose 6-phosphate, followed by immunoprecipitation of the column fractions, we identified the putative MPR 46 protein in the Bg cells. When Bg cell MPR 46 was deglycosylated along with chicken MPR 46 (control) both species yielded a single polypeptide corresponding to molecular mass of 26 kDa, suggesting that both contain the same receptor protein.