A new bile acid conjugate, ciliatocholic acid, from bovine gall bladder bile. (1/26)

This study was carried out to investigate the occurrence of ciliatocholic acid in bovine gall bladder bile. Ciliatocholic acid was synthesized according to the method described by Bergstrom and Norman for the synthesis of taurocholic acid. Elemental analysis, melting point, and the infrared spectrum of this substance were determined. An isolation procedure for ciliatocholic acid was established by stepwise elution with an HCl-ethanol solvent system using a Dowex-1 anion exchange resin column chromatographic technique. Ciliatocholic acid amounting to 158 mug (as ciliatine) per 100 ml of gall bladder bile was found in the fraction eluted with 0.01 N HCl in 50% ethanol. This coumpound was purified by preparative thin-layer chromatography and confirmed to be ciliatocholic acid from the hydrolytic stability, phosphorus determination, and chromatographic behavior. Thus, bovine gall bladder bile contains a small amount of ciliatocholic acid.  (+info)

Aromatic L-amino acid decarboxylase: conformational change in the flexible region around Arg334 is required during the transaldimination process. (2/26)

Aromatic L-amino acid decarboxylase (AADC) catalytic mechanism has been proposed to proceed through two consecutive intermediates (i.e., Michaelis complex and the external aldimine). Limited proteolysis of AADC that preferentially digested at the C-terminal side of Arg334 was slightly retarded in the presence of dihydroxyphenyl acetate that formed a stable Michaelis complex. On the contrary, AADC was scarcely digested in the presence of L-dopa methyl ester that formed a stable external aldimine. Similar protection by the substrate analogs was observed in the chemical modification experiment. From these results, we concluded that the region around Arg334 must be exposed and flexible in the unliganded state, and forming the Michaelis complex generated a subtle conformational change, then underwent marked conformational change during the subsequent transaldimination process prerequisite to forming the external aldimine. For further analyses, we constructed a mutant gene encoding in tandem the two peptides of AADC cleaved at the Asn327-Met328 bond inside the putative flexible region. The gene product, fragmentary AADC, was still active with L-dopa as substrate, but its k(cat) value was decreased 57-fold, and the Km value was increased 9-fold compared with those of the wild-type AADC. The absorption spectra of the fragmentary AADC in the presence of L-dopa methyl ester showed shift in the equilibrium of the transaldimination from the external aldimine to the Michaelis complex. Tryptic digestion of the fragmentary AADC removed seven amino acid residues, Met328-Arg334, and resulted in complete inactivation. Susceptibility of the fragmentary enzyme to trypsin was not changed by L-dopa methyl ester revealing the loss of appropriate conformational change in the flexible region induced by substrate binding. From these results we propose that the conformational change in the flexible region is required during the transaldimination process.  (+info)

Structural determinants for ligand binding and catalysis of triosephosphate isomerase. (3/26)

The crystal structure of leishmania triosephosphate isomerase (TIM) complexed with 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate (IPP) highlights the importance of Asn11 for binding and catalysis. IPP is an analogue of the substrate D-glyceraldehyde-3-phosphate, and it is observed to bind with its aldehyde oxygen in an oxyanion hole formed by ND2 of Asn11 and NE2 of His95. Comparison of the mode of binding of IPP and the transition state analogue phosphoglycolohydroxamate (PGH) suggests that the Glu167 side chain, as well as the triose part of the substrate, adopt different conformations as the catalysed reaction proceeds. Comparison of the TIM-IPP and the TIM-PGH structures with other liganded and unliganded structures also highlights the conformational flexibility of the ligand and the active site, as well as the conserved mode of ligand binding.  (+info)

The 2-aminoethylphosphonate-specific transaminase of the 2-aminoethylphosphonate degradation pathway. (4/26)

The 2-aminoethylphosphonate transaminase (AEPT; the phnW gene product) of the Salmonella enterica serovar Typhimurium 2-aminoethylphosphonate (AEP) degradation pathway catalyzes the reversible reaction of AEP and pyruvate to form phosphonoacetaldehyde (P-Ald) and L-alanine (L-Ala). Here, we describe the purification and characterization of recombinant AEPT. pH rate profiles (log V(m) and log V(m)/K(m) versus pH) revealed a pH optimum of 8.5. At pH 8.5, K(eq) is equal to 0.5 and the k(cat) values of the forward and reverse reactions are 7 and 9 s(-1), respectively. The K(m) for AEP is 1.11 +/- 0.03 mM; for pyruvate it is 0.15 +/- 0.02 mM, for P-Ald it is 0.09 +/- 0.01 mM, and for L-Ala it is 1.4 +/- 0.03 mM. Substrate specificity tests revealed a high degree of discrimination, indicating a singular physiological role for the transaminase in AEP degradation. The 40-kDa subunit of the homodimeric enzyme is homologous to other members of the pyridoxalphosphate-dependent amino acid transaminase superfamily. Catalytic residues conserved within well-characterized members are also conserved within the seven known AEPT sequences. Site-directed mutagenesis demonstrated the importance of three selected residues (Asp168, Lys194, and Arg340) in AEPT catalysis.  (+info)

Properties of phosphoenolpyruvate mutase, the first enzyme in the aminoethylphosphonate biosynthetic pathway in Trypanosoma cruzi. (5/26)

Phosphoenolpyruvate (PEP) mutase catalyzes the conversion of phosphoenolpyruvate to phosphonopyruvate, the initial step in the formation of many naturally occurring phosphonate compounds. The phosphonate compound 2-aminoethylphosphonate is present as a component of complex carbohydrates on the surface membrane of many trypanosomatids including glycosylinositolphospholipids of Trypanosoma cruzi. Using partial sequence information from the T. cruzi genome project we have isolated a full-length gene with significant homology to PEP mutase from the free-living protozoan Tetrahymena pyriformis and the edible mussel Mytilus edulis. Recombinant expression in Escherichia coli confirms that it encodes a functional PEP mutase with a Km apparent of 8 microM for phosphonopyruvate and a kcat of 12 s-1. The native enzyme is a homotetramer with an absolute requirement for divalent metal ions and displays negative cooperativity for Mg2+ (S0.5 0.4 microM; n = 0.46). Immunofluorescence and sub-cellular fractionation indicates that PEP mutase has a dual localization in the cell. Further evidence to support this was obtained by Western analysis of a partial sub-cellular fractionation of T. cruzi cells. Southern and Western analysis suggests that PEP mutase is unique to T. cruzi and is not present in the other medically important parasites, Trypanosoma brucei and Leishmania spp.  (+info)

Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system. (6/26)

The plant pathogen Agrobacterium tumefaciens forms architecturally complex biofilms on inert surfaces. Adherence of A. tumefaciens C58 was significantly enhanced under phosphate limitation compared to phosphate-replete conditions, despite slower overall growth under low-phosphate conditions. Replacement of Pi with sn-glycerol-3-phosphate and 2-aminoethylphosphonate yielded similar results. The increase in surface interactions under phosphate limitation was observed in both static culture and continuous-culture flow cells. Statistical analysis of confocal micrographs obtained from the flow cell biofilms revealed that phosphate limitation increased both the overall attached biomass and the surface coverage, whereas the maximum thickness of the biofilm was not affected. Functions encoded on the two large plasmids of A. tumefaciens C58, pTiC58 and pAtC58, were not required for the observed phosphate effect. The phosphate concentration at which increased attachment was observed triggered the phosphate limitation response, controlled in many bacteria by the two-component regulatory system PhoR-PhoB. The A. tumefaciens phoB and phoR orthologues could only be disrupted in the presence of plasmid-borne copies of the genes, suggesting that this regulatory system might be essential. Expression of the A. tumefaciens phoB gene from a tightly regulated inducible promoter, however, correlated with the amount of biofilm under both phosphate-limiting and nonlimiting conditions, demonstrating that components of the Pho regulon influence A. tumefaciens surface interactions.  (+info)

Utilization of 2-aminoethylarsonic acid in Pseudomonas aeruginosa. (7/26)

This paper describes the metabolism, transport and growth inhibition effects of 2-aminoethylarsonic acid (AEA) and 3-aminopropylarsonic acid (APrA). The former compound supported growth of Pseudomonas aeruginosa, as sole nitrogen source. The two arsonates inhibited the growth of this bacterium when 2-aminoethylphosphonic acid (AEP) but not alanine or NH4Cl, was supplied as the only other nitrogen source. The analogy between AEA and the natural compound AEP led us to examine the in vitro and in vivo interaction of AEA with the enzymes of AEP metabolism. The uptake system for AEP (Km 6 microM) was found to be competitively inhibited by AEA and APrA (Ki 18 microM for each). AEP-aminotransferase was found to act on AEA with a Km of 4 mM (3.85 mM for AEP). Alanine and 2-arsonoacetaldehyde was generated concomitantly, in a stoichiometric reaction. In vivo, AEA was catabolized by the AEP-aminotransferase since it was able to first induce this enzyme, then to be an efficient substrate. The lower growth observed may have been due to the slowness with which the permease and the aminotransferase were induced, and hence to a poor supply of alanine by transamination.  (+info)

Reversible phase variation in the phnE gene, which is required for phosphonate metabolism in Escherichia coli K-12. (8/26)

It is known that Escherichia coli K-12 is cryptic (Phn-) for utilization of methyl phosphonate (MePn) and that Phn+ variants can be selected for growth on MePn as the sole P source. Variants arise from deletion via a possible slip strand mechanism of one of three direct 8-bp repeat sequences in phnE, which restores function to a component of a putative ABC type transporter. Here we show that Phn+ variants are present at the surprisingly high frequency of >10(-2) in K-12 strains. Amplified-fragment length polymorphism analysis was used to monitor instability in phnE in various strains growing under different conditions. This revealed that, once selection for growth on MePn is removed, Phn+ revertants reappear and accumulate at high levels through reinsertion of the 8-bp repeat element sequence. It appears that, in K-12, phnE contains a high-frequency reversible gene switch, producing phase variation which either allows ("on" form) or blocks ("off" form) MePn utilization. The switch can also block usage of other metabolizable alkyl phosphonates, including the naturally occurring 2-aminoethylphosphonate. All K-12 strains, obtained from collections, appear in the "off" form even when bearing mutations in mutS, mutD, or dnaQ which are known to enhance slip strand events between repetitive sequences. The ability to inactivate the phnE gene appears to be unique to K-12 strains since the B strain is naturally Phn+ and lacks the inactivating 8-bp insertion in phnE, as do important pathogenic strains for which genome sequences are known and also strains isolated recently from environmental sources.  (+info)