Characterization of the cell surface protein gene of Corynebacterium ammoniagenes.

Three dominant cell surface proteins of Corynebacterium ammoniagenes ATCC 6872 were identified in the cell wall fraction. The cspA gene, which encodes one of the major cell surface proteins, was cloned using the N-terminal amino acid sequence of the protein. Then the cloned chromosomal fragment containing the cspA gene was sequenced and was shown to encode a mature polypeptide of 333 amino acids with a molecular mass of 36654 Da. The amino acid sequence of the cspA gene showed similarity to the amino acid sequence of C. glutamicum CspA, one of the two major secreted proteins of C. glutamicum, although C. ammoniagenes CspA and C. glutamicum CspA differed in size. Northern blot analysis and primer extension analysis respectively revealed a 1.1 kb transcript and a promoter sequence resembling that of the C. ammoniagenes fatty acid synthase B (fasB) gene.

Acid phosphatase/phosphotransferases from enteric bacteria.

We have investigated the enzymatic phosphorylation of nucleosides and found that Morganella morganii phoC acid phosphatase exhibits regioselective pyrophosphate (PP(i))-nucleoside phosphotransferase activity. In this study, we isolated genes encoding an acid phosphatase with regioselective phosphotransferase activity (AP/PTase) from Providencia stuartii, Enterobacter aerogenes, Escherichia blattae and Klebsiella planticola, and compared the primary structures and enzymatic characteristics of these enzymes with those of AP/PTase (PhoC acid phosphatase) from M. morganii. The enzymes were highly homologous in primary structure with M. morganii AP/PTase, and are classified as class A1 acid phosphatases. The synthesis of inosine-5'-monophosphate (5'-IMP) by E. coli overproducing each acid phosphatase was investigated. The P. stuartii enzyme, which is most closely related to the M. morganii enzyme, exhibited high 5'-IMP productivity, similar to the M. morganii enzyme. The 5'-IMP productivities of the E. aerogenes, E. blattae and K. planticola enzymes were inferior to those of the former two enzymes. This result underlines the importance of lower K(m) values for efficient nucleotide production. As these enzymes exhibited a very high degree of homology at the amino acid sequence level, it is likely that local sequence differences in the binding pocket are responsible for the differences in the nucleoside-PP(i) phosphotransferase reaction.

Phosphorylation of guanosine using guanosine-inosine kinase from Exiguobacterium acetylicum coupled with ATP regeneration.

Guanosine 5'-monophosphate (5'-GMP) and inosine 5'-monophosphate (5'-IMP) are widely used as flavor enhancers. Recently, a novel process for 5'-IMP production by phosphorylation of inosine using guanosine-inosine kinase coupled with ATP regeneration was reported. In this study, we demonstrated the practical possibility of producing 5'-GMP by phosphorylation of guanosine using a guanosine-inosine kinase from Exiguobacterium acetylicum coupled with ATP regeneration.

End-product regulation and kinetic mechanism of guanosine-inosine kinase from Escherichia coli.

Escherichia coli guanosine-inosine kinase was overproduced, purified, and characterized. The native and subunit molecular weights were 85,000 and 45,000, respectively, indicating that the enzyme was a dimer. A pI of 6.0 was obtained by isoelectric focusing. In addition to ATP, it was found that deoxyadenosine 5'-triphosphate, UTP, and CTP could serve as phosphate donors. The phosphate acceptors were guanosine, inosine, deoxyguanosine and xanthosine, but not adenosine, cytidine, uridine, or deoxythymidine. Maximum activity was attained at an ATP/Mg2+ concentration ratio of 0.5. In the presence of pyrimidine nucleotides, enzyme activity was slightly increased, while it was markedly inhibited by GDP and GTP. Initial velocity and product inhibition studies support an ordered Bi Bi mechanism in which guanosine was the first substrate to bind and GMP was the last product to be released. Guanosine kinase may be a regulatory enzyme that has a role in modulating nucleotide levels.

Phosphorylation of nucleosides by the mutated acid phosphatase from Morganella morganii.

A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. The Morganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to the M. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced into Escherichia coli, and the transformants were screened for the production of 5'-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5'-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreased K(m) value for inosine was responsible for the increased productivity.

Development of an improved assay for purine nucleoside kinase activity in cell extracts and detection of inosine kinase activity in Brevibacterium acetylicum ATCC 953, related species, and Corynebacterium flaccumfaciens ATCC 6887.

An improved assay was developed to detect direct purine nucleoside phosphorylating activity in cell-free extracts. Direct inosine phosphorylating activity was detected in 2 of 70 species tested. Both activities, which depended on magnesium ion and ATP, phosphorylated a hydroxyl group at the 5' position of inosine. The new assay was shown to be useful for screening of direct purine nucleoside phosphorylating activity and have the potential to detect inosine kinase in the presence of a background of nucleoside phosphorylase and purine phosphoribosyltransferase activities. Previously, the latter two activities made it difficult to correctly detect direct phosphorylation of inosine by inosine kinase.

Molecular characterization of guanosine kinase gene from a facultative alkalophile, Exiguobacterium aurantiacum ATCC 35652.

Guanosine kinase activity was identified in an alkalophile, Exiguobacterium aurantiacum ATCC 35652, and the gsk gene encoding it was isolated as a homologous fragment of the Brevibacterium acetylicum gsk. The cloned chromosomal fragment contained an open reading frame which encodes a polypeptide of 308 amino acids with a molecular mass of 33349 Da. The Ex. aurantiacum Gsk shows strong sequence similarity to B. acetylicum Gsk, but possesses five additional amino acids at the C-terminus. An Mr 39200 protein synthesized in Escherichia coli harboring plasmids carrying Ex. aurantiacum gsk was purified and characterized. A 0.9 kb transcript revealed by Northern blot analysis and the presence of a factor-independent terminator structure suggest that Ex. aurantiacum gsk is monocistronic, although B. acetylicum gsk has been shown to be bicistronic. The striking alterations in the 3'-untranslated region resulted in a different gene organization.

Molecular cloning and transcriptional analysis of a guanosine kinase gene of Brevibacterium acetylicum ATCC 953.

The Brevibacterium acetylicum gsk gene, which encodes guanosine kinase (ATP:guanosine 5'-phosphotransferase), a kinase that is involved in guanosine salvage pathways, has been cloned by using the N-terminal amino acid sequence of the purified protein. The cloned chromosomal fragment containing the gsk gene was sequenced and shown to encode a polypeptide of 303 amino acids with a molecular mass of 32,536 Da, which is in good agreement with the measured molecular weight of the purified enzyme. Recombinant Escherichia coli strains harboring plasmids carrying the B. acetylicum gsk gene overexpressed both guanosine and inosine kinase activities. The primary structure of the gsk gene shows similarity to amino acid sequences of sugar kinases classified in the ribokinase family stronger than to those of the E. coli gsk gene encoding guanosine kinase and other nucleoside kinases. Northern blot analysis and primer extension analysis revealed a 1.4-kb transcript and promoter sequences, like the E. coli sigma70 and B. subtilis sigmaA consensus sequences, respectively. These results, together with the nucleotide sequence of the downstream region of gsk, suggested that the organization of B. acetylicum gsk is bicistronic. The second gene, orf2, shows significant similarity to the mutT mutator genes of several organisms, although its function has not yet been identified. The gsk gene was specifically transcribed in the early exponential growth phase, which seems to correspond to the specific guanosine kinase activity profile and suggests a role in controlling the nucleoside monophosphate level by efficiently recycling guanosine when cells are in the early exponential phase.

Characterization of guanosine kinase from Brevibacterium acetylicum ATCC 953.

Guanosine kinase (GKase) activity was identified in a cell-free extract of Brevibacterium acetylicum ATCC 953. We have purified the enzyme 4000-fold from the cell-free extract to apparent homogeneity. Molecular weight of 71,300 and 36,300 determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively, revealed that the enzyme is a dimer molecule. Maximum activity of the GKase was attained when the magnesium/ATP concentration ratio was close to 1. The GKase activity was dependent on the presence of a divalent cation. The Km values for guanosine, inosine, and 2'-deoxyguanosine as phosphate acceptors were determined to be 0.022, 0.87, and 2.83 mM, respectively. In addition to ATP and dATP, GTP and dGTP were shown to be effective phosphate donors for the GKase. The optimal pH seemed to be around pH 8.3, although relatively high activity was observed in the alkaline pH range of 7.5-9.8. The addition of 0.1 mM pyrimidine nucleotides, especially CMP and CTP, activated the GKase activity. On the other hand, the addition of 1 mM AMP, ADP, and GMP inhibited the GKase activity. It is thus likely that the GKase activity might be regulated in vivo by nucleotide concentrations and may control the nucleoside monophosphate level by efficiently recycling guanosine.

Epstein-Barr virus-associated malignant lymphoma with macroamylasemia and monoclonal gammopathy in a patient with Wiskott-Aldrich syndrome.

A 1-year-old boy with Wiskott-Aldrich Syndrome (WAS) who developed malignant lymphoma is described. He showed various complications such as atypical lymphocytosis, disseminated intravascular coagulation (DIC), intracranial hemorrhage, macroamylasemia, and monoclonal gammopathy (immunoglobulin A kappa chain). Epstein-Barr virus (EBV) DNA was detected in the tumor tissue, and the monoclonality of B cells from the tumor tissue was established. EBV-associated lymphoma is frequently observed in immunocompromised patients including those with WAS. The development of macroamylasemia, which is rare in childhood, is discussed in relation to lymphoma and monoclonal gammopathy. This case is unique in that the EBV-associated malignant lymphoma developed at an early age and was accompanied by macroamylasemia.

Syntheses of L-tyrosine-related amino acids by tyrosine phenol-lyase of Citrobacter intermedius.

Degradation of tyrosine to phenol, pyruvate and ammonia by tyrosine phenol-lyase from Citrobacter intermedius (formerly named Escherichia intermedia) is readily reversible at high concentrations of pyruvate and ammonia. Spectrophotometric studies indicate that ammonia is the first substrate which interacts with bound pyridoxal 5'-phosphate. Kinetic results show that pyruvate is the second substrate bound, hence phenol must be the third. When an appropriate phenol derivative is substituted for phenol, the corresponding tyrosine analogue can be synthesized. 3-Fluoro-, 2-fluoro-, 3-chloro-, 2-chloro-, 3-bromo-, 2-bromo-, 2-iodo-, 3-methyl-, 2-methyl- and 2-methoxy-L-tyrosines have been synthesized by this reaction. By using various phenol derivatives or tyrosine analogues as substrates, the substrate specificity of tyrosine phenol-lyase is investigated and the situation of its active site is discussed.

A new and facile synthesis of purine 2'-amino-2'-deoxyribosides by a combination of chemical and enzymatic reactions.

An enzymatic synthesis of 2'-amino-2'-deoxy-2-chloroinosine and its chemical conversion to purine 2'-amino-2'-deoxyribosides are described. In addition, some of 2'-amino-2'-deoxy-ribosides of 6-substituted purine were also prepared from 2'-amino-2'-deoxyinosine.

A new synthesis of purine arabinosides by a combination of chemical and enzymatic reaction.

The synthesis of hypoxanthine, 2-chlorohypoxanthine, and 2-methylhypoxanthine arabinoside by an enzymatic transarabinosylation and their chemical conversion to biologically interesting purine arabinosides are described. Some of the synthesized compounds were tested for the inhibition of DNA synthesis in cultured tumor cells.

Studies on tyrosine phenol lyase. Modification of essential histidyl residues by diethylpyrocarbonate.

Tyrosine phenol-lyase of Escherichia intermedia is inactivated by treatment with diethylpyrocarbonate at pH 6.0 AND 4 degrees. Spectrophotometric studies show that the inactivation is stoichiometric, with a modification of 2 histidyl residues per molecule of the enzyme. Finding that this inactivation is largely reversed by treatment with hydroxylamine indicates that the inactivation is mainly due to modification of the histidyl residues. No changes in the sulfhydryl content or in the aromatic amino acids are observed as a result of this modification. The modified tyrosine phenol-lyase retains most of its ability to form a nearly normal complex with its coenzyme, pyridoxal phosphate. This has been shown by studies of its absorption, by the determination of pyridoxal phosphate, and by reduction of the holoenzyme with tritiated sodium borohydride. The modified enzyme also appears to form a Schiff base intermediate with L-alanine. The modified holoenzyme fails to catalyze the exchange of the alpha-hydrogen of L-alanine with tritium from tritiated water. This is consistent with a catalytic role for modified histidyl residues at the active site of the enzyme; this role is the removal of the alpha-hydrogen of substrates.