Phosphorylation of Phenol by Phenylphosphate Synthase: Role of Histidine Phosphate in Catalysis

ABSTRACT The anaerobic metabolism of phenol proceeds via carboxylation to 4-hydroxybenzoate by a two-step process involving seven proteins and two enzymes (“biological Kolbe-Schmitt carboxylation”). MgATP-dependent phosphorylation of phenol catalyzed by phenylphosphate synthase is followed by phenylphosphate carboxylation. Phenylphosphate synthase shows similarities to phosphoenolpyruvate (PEP) synthase and was studied for the bacterium Thauera aromatica. It consists of three proteins and transfers the β-phosphoryl from ATP to phenol; the products are phenylphosphate, AMP, and phosphate. We showed that protein 1 becomes phosphorylated in the course of the reaction cycle by [β-32P]ATP. This reaction requires protein 2 and is severalfold stimulated by protein 3. Stimulation of the reaction by 1 M sucrose is probably due to stabilization of the protein(s). Phosphorylated protein 1 transfers the phosphoryl group to phenolic substrates. The primary structure of protein 1 was analyzed by nanoelectrospray mass spectrometry after CNBr cleavage, trypsin digestion, and online high-pressure liquid chromatography at alkaline pH. His-569 was identified as the phosphorylated amino acid. We propose a catalytic ping-pong mechanism similar to that of PEP synthase. First, a diphosphoryl group is transferred to His-569 in protein 1, from which phosphate is cleaved to render the reaction unidirectional. Histidine phosphate subsequently serves as the actual phosphorylation agent.

Identification of a novel 45 kDa protein (JP-45) from rabbit sarcoplasmic-reticulum junctional-face membrane

Using a biochemical/immunological approach to analyse the protein constituents of skeletal-muscle junctional-face membrane (JFM), we identified a 45kDa protein. Its N-terminal amino acid was blocked, but the amino acid sequence obtained from several peptides after proteolytic treatment did not significantly match that of any protein present in the SwissProt and NCBI (National Center for Biotechnology Information) databases. We synthesized a peptide whose sequence matched that of one of the peptides obtained after CNBr cleavage of the 45kDa protein; the peptide was conjugated to a carrier and used to raise antibodies. The antiserum was used to study in more detail the biochemical characteristics of the novel 45kDa protein. Analysis of the proteins present in different subcellular membrane fractions show that the novel 45kDa polypeptide: (i) is an integral membrane constituent present both in neonatal and adult skeletal-muscle sarcoplasmic reticulum; (ii) is selectively localized in the JFM; (iii) is not present in microsomes obtained from rabbit heart, liver or kidney. Immunoprecitation with anti-(45kDa protein) antibody indicates that the 45kDa protein is part of a complex which can be phosphorylated in vitro by the catalytic subunit of protein kinase A.

Three spinach leaf nitrate reductase-3-hydroxy-3-methylglutaryl-CoA reductase kinases that are regulated by reversible phosphorylation and/or Ca2+ ions

In spinach (Spinacea oleracea L.) leaf extracts, three protein kinases (PKI, PKII and PKIII) were identified each of which phosphorylated spinach nitrate reductase on serine-543, and inactivated the enzyme in the presence of nitrate reductase inhibitor, 14-3-3. PKIII was also very active in phosphorylating and inactivating Arabidopsis(Landsberg erecta) 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMGR1). PKI and PKII phosphorylated HMGR1 more slowly than PKIII, compared with their relative rates of phosphorylation of nitrate reductase. All three kinases gave phosphopeptide CNBr-cleavage maps of HMGR1 identical with those that are seen after phosphorylation of serine-577 by the sucrose non-fermenting (SNF1)-like PK, 3-hydroxy-3-methylglutaryl-Co A reductase kinase A (HRK-A), from cauliflower [Dale, Arró, Becerra, Morrice, Boronat, Hardie and Ferrer (1995) Eur. J. Biochem. 233, 506–513]. PKI was Ca2+-dependent when prepared in the absence of protein phosphatase (PP) inhibitors, and largely Ca2+-dependent when prepared in the presence of PP inhibitors (NaF and EGTA). The Ca2+-independent portion of PKI was inactivated by either PP2A or PP2C, while the Ca2+-dependent portion of PKI became increasingly activated during storage, which we presume was mimicking the effect of an unidentified PP. These findings indicate that PKI is regulated by two functionally distinct phosphorylations. PKI had a molecular mass of 45 kDa on gel filtration and was active towards substrate peptides that terminated at the +2 residue from the phosphorylation site, whereas PKIII was inactive towards these peptides. PKII was Ca2+-stimulated under all conditions tested. PKIII was Ca2+-independent, inactivated by PP2A or PP2C, had a requirement for a hydrophobic residue in the +4 position of peptide substrates, had a molecular mass by gel filtration of ∼140 kDa, and an antibody against the rye SNF1-related PK (RKIN1) recognised a 58 kDa subunit in fractions containing PKIII. These properties of PKIII are identical with those reported previously for the SNF1-like enzyme, HRK-A. Our results indicate a considerable complexity of kinase cascades mediating the regulation of assimilatory and biosynthetic pathways in response to environmental stimuli in plants.

Expression and epitopic conservation of calponin in different smooth muscles and during development

Calponin is a thin filament associated protein found in smooth muscle as a potential modulator of contraction. Five mouse monoclonal antibodies (mAbs CP1, CP3, CP4, CP7, and CP8) were prepared against chicken gizzard α-calponin. The CP1 epitopic structure is conserved in smooth muscles across vertebrate phyla and is highly sensitive to CNBr cleavage in contrast with the chicken-specific CP4 and the avian–mammalian-specific CP8 epitopes that are resistant to CNBr fragmentation. Using this panel of mAbs against multiple epitopes, only α-calponin was detected in adult chicken smooth muscles and throughout development of the gizzard. Western blotting showed that the calponin content varied among different smooth muscle tissues and correlated with that of h-caldesmon. In contrast with the constitutive expression of calponin in phasic smooth muscle of the digestive tract, very low levels of calponin were detected in adult avian tracheas and no calponin expression was detected in embryonic and young chick tracheas. These results provide information on the structural conservation of calponins and suggest a relationship between calponin expression and smooth muscle functional states.Key words: smooth muscle calponin, caldesmon, expression, development, chicken trachea.

Purification and characterization of a novel phosphoenolpyruvate carboxylase from banana fruit

Phosphoenolpyruvate carboxylase (PEPC) from ripened banana (Musa cavendishii L.) fruits has been purified 127-fold to apparent homogeneity and a final specific activity of 32 mumol of oxaloacetate produced/min per mg of protein. Non-denaturing PAGE of the final preparation resolved a single protein-staining band that co-migrated with PEPC activity. Polypeptides of 103 (alpha-subunit) and 100 (beta-subunit) kDa, which stain for protein with equal intensity and cross-react strongly with anti-(maize leaf PEPC) immune serum, were observed following SDS/PAGE of the final preparation. CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. The enzyme's native molecular mass was estimated to be about 425 kDa. These data indicate that in contrast to the homotetrameric PEPC from most other sources, the banana fruit enzyme exists as an alpha 2 beta 2 heterotetramer. Monospecific rabbit anti-(banana PEPC) immune serum effectively immunoprecipitated the activity of the purified enzyme. Immunoblotting studies established that the 100 kDa subunit did not arise via proteolysis of the 103 kDa subunit after tissue extraction, and that the subunit composition of banana PEPC remains uniform throughout the ripening process. PEPC displayed a typical pH activity profile with an alkaline optimum and activity rapidly decreasing below pH 7.0. Enzymic activity was absolutely dependent on the presence of a bivalent metal cation, with Mg2+ or Mn2+ fulfilling this requirement. The response of the PEPC activity to PEP concentration and to various effectors was greatly influenced by pH and glycerol addition to the assay. The enzyme was activated by hexose-monophosphates and potently inhibited by malate, succinate, aspartate and glutamate at pH 7.0, whereas the effect of these metabolites was considerably diminished or completely abolished at pH 8.0. The significance of metabolite regulation of PEPC is discussed in relation to possible functions of this enzyme in banana fruit metabolism.