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A T Phillips

Publications and source records attributed to A T Phillips.

At least 37 records · Page 2Linked to original sources
PubMed1988

Organization and multiple regulation of histidine utilization genes in Pseudomonas putida.

The arrangement of the histidine utilization (hut) genes in Pseudomonas putida was established by examining the structure of a DNA segment that had been cloned into Escherichia coli via a cosmid vector. Southern blot analysis revealed that the restriction patterns of the hut genes cloned into E. coli and present in the P. putida genome were identical, indicating that no detectable DNA rearrangement took place during the cloning. Expression of the hut genes from a series of overlapping clones indicated the gene order to be hutG-hutI-hutH-hutU-hutC-hutF. The transcription directions of the different hut genes were determined by cloning the genes under control of the lambda pL promoter. This showed that hutF, encoding formiminoglutamate hydrolase, was transcribed in a direction opposite to that of the other genes. Inactivation of the cloned hut genes by Tn1000 insertion revealed that the hut genes were divided into three major transcriptional units (hutF, hutC [the repressor gene], and hut UHIG), but hutG may also be independently transcribed. When cloned individually with hutC on the same vector, hutF and hutU (which encodes urocanase) expression was induced by urocanate, indicating that these two genes each possess an operator-promoter element. Tn1000 insertions (in the cloned genes) or Tn5 insertions (in the P. putida genome) affecting the hutI or hutH gene only partially eliminated hutG expression. Furthermore, hutG, which specifies N-formylglutamate amidohydrolase, was regulated by the hutC product when the two genes were cloned on the same vector and expressed in E. coli. Therefore, hutG can be expressed independently from its own promoter, in keeping with earlier observations that N-formylglutamate amidohydrolase synthesis is not coordinated with that of urocanase and histidase and can be induced by N-formylglutamate or urocanate.

Cloning, Molecular↗
PubMed1987

Purification and properties of formylglutamate amidohydrolase from Pseudomonas putida.

Formylglutamate amidohydrolase (FGase) catalyzes the terminal reaction in the five-step pathway for histidine utilization in Pseudomonas putida. By this action, N-formyl-L-glutamate (FG) is hydrolyzed to produce L-glutamate plus formate. Urocanate, the first product in the pathway, induced all five enzymes, but FG was able to induce FGase alone, although less efficiently than urocanate did. This induction by FG resulted in the formation of an FGase with electrophoretic mobility identical to that of the FGase induced by urocanate. A 9.6-kilobase-pair HindIII DNA fragment containing the P. putida FGase gene was cloned into the corresponding site on plasmid pBEU1 maintained in Escherichia coli. Insertion of the fragment in either orientation on the vector resulted in expression, but a higher level was noted in one direction, suggesting that the FGase gene can be expressed from either of two vector promoters with different efficiencies or from a single vector promoter in addition to a less efficient Pseudomonas promoter. FGase was purified 1,110-fold from the higher-expression clone in a yield of 10% through six steps. Divalent metal ions stimulated activity, and among those tested (Co, Fe, Zn, Ca, Ni, Cd, Mn, and Mg), Co(II) was the best activator, followed by Fe(II). FGase exhibited a Km of 14 mM for FG and a specific activity of 100 mumol/min per mg of protein in the presence of 5 mM substrate and 0.8 mM CoCl2 at 30 degrees C. The enzyme was maximally active in the range of pH 7 to 8. FGase was found to be a monomer of molecular weight 50,000. N-Acetyl-L-glutamate was not a substrate for the enzyme, but both it and N-formyl-L-aspartate were competitive inhibitors of formylglutamate hydrolysis, exhibiting Ki values of 6 and 9 mM, respectively. The absence of FGase activity as an integral part of histidine breakdown in most other organisms and the somewhat uncoordinated regulation of FGase synthesis with that of the other hut enzymes in Pseudomonas suggest that the gene encoding its synthesis may have evolved separately from the remaining hut genes.

Amidohydrolases↗
PubMed1985

Presence and quantity of dehydroalanine in histidine ammonia-lyase from Pseudomonas putida.

Dehydroalanine is present in the histidine ammonia-lyase (histidase) from Pseudomonas putida ATCC 12633 as shown by reaction of purified enzyme with K14CN or NaB3H4 and subsequent identification of [14C]aspartate or [3H]alanine, respectively, following acid hydrolysis of the labeled protein. When labeling with cyanide was conducted under denaturing conditions, 4 mol of [14C]cyanide was incorporated per mol of enzyme (Mr 220 000), equivalent to one dehydroalanine residue being modified per subunit in this protein composed of four essentially identical subunits. In native enzyme, inactivation of catalytic activity by cyanide was complete when 1 mol of [14C]cyanide had reacted per mol of histidase, suggesting that modification of any one of the four dehydroalanine residues in the tetrameric enzyme was sufficient to prevent catalysis at all sites. Loss of activity on treatment with cyanide could be blocked by the addition of the competitive inhibitor cysteine or substrate if Mn2+ was also present. Cross-linking of native enzyme with dimethyl suberimidate produced no species larger than tetramer, thereby eliminating the possibility that an aggregation phenomenon might explain why only one-fourth of the dehydroalanyl residues was modified by cyanide during inactivation. A labeled tryptic peptide was isolated from enzyme inactivated with [14C]cyanide. Its composition was different from that of a tryptic peptide previously isolated from other histidases and shown to contain a highly reactive and catalytically important cysteine residue. Such a finding indicates the dehydroalanine group is distinct from the active site cysteine. Treatment of crude extracts with [14C]cyanide and purification of the inactive enzyme yielded labeled protein that release [14C]aspartate on acid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1985

Cloning and expression in Escherichia coli of histidine utilization genes from Pseudomonas putida.

A library of the Pseudomonas putida chromosome, prepared through the use of the cosmid pJB8 ligated to a partial Sau3A digest of bacterial DNA, followed by in vitro packaging into bacteriophage lambda particles, was used to construct a strain of Escherichia coli which contained the genes for histidine utilization. This isolate produced a repressor product and all five enzymes required in Pseudomonas spp. for histidine dissimilation, whereas none of these could be detected in the nontransduced parent E. coli strain. When this transductant was grown on various media containing histidine or urocanate as the inducer, it was observed that production of the cloned histidine degradative enzymes was influenced somewhat by the choice of nitrogen source used but not by the carbon source. The recombinant cosmid was isolated and found to consist of 21.1 kilobase pairs of DNA, with approximately 16 kilobase pairs derived from Pseudomonas DNA and the remainder being from the pJB8 vector. Digestion of this insert DNA with EcoRI provided a 6.1-kilobase-pair fragment which, upon ligation in pUC8 and transformation into an E. coli host, was found to encode histidine ammonia-lyase and urocanase. The inducible nature of this production indicated that the hut repressor gene also was present on this fragment. Insertional inactivation of the histidine ammonia-lyase and urocanase genes by the gamma-delta transposon has permitted location of these structural genes and has provided evidence that transcription proceeds from urocanase through histidine ammonia-lyase. Mapping of the 16-kilobase-pair Pseudomonas DNA segment with restriction enzymes and subcloning of additional portions, one of which contained the gene for formiminoglutamate hydrolase and another that could constitutively express activities for both imidazolone propionate hydrolase and formylglutamate hydrolase, has provided evidence for the organization of all hut genes.

Cloning, Molecular↗
PubMed1983

Identification of the enol tautomer of imidazolone propionate as the urocanase reaction product.

A fluorescent product was transiently formed during catalysis by urocanase from Pseudomonas putida. The fluorophore showed an emission maximum at 430 nm when excited at 330 nm, essentially identical to that exhibited by the enol tautomer of imidazolone propionate. The keto isomer was not fluorescent under these conditions. In aqueous acid solutions where imidazolone propionate is relatively stable, an equilibrium mixture of tautomeric forms contained approximately 1% of the enol isomer. In ethanolic solutions, the equilibrium concentration of enol tautomer increased to approximately 25%. The differing content of imidazolone propionate tautomers as a function of solvent conditions permitted a comparison of the keto and enol forms as substrates for the reverse reaction. This revealed an almost complete preference for the enol tautomer. These results are taken as direct proof that enol imidazolone propionate is the true urocanase reaction product.

Chemical Phenomena↗
PubMed1981

Mechanism of urocanase as studied by deuterium isotope effects and labeling patterns.

Nicotinamide adenine dinucleotide (NAD) dependent urocanase (4'-imidazolone-5'-propionate hydro-lyase, EC 4.2.1.49) from Pseudomonas putida was found to catalyze an exchange reaction between solvent and the 4'-hydrogen of urocanate or imidazolepropionate at a rate faster than that of overall deuterium was compared to unlabeled urocanate as a substrate, no isotope rate effect was noted. For examination of the possibility of an NAD+-mediated intramolecular hydride transfer of the 4'-hydrogen to a position on the side chain of oxoimidazolepropionate, the origins of hydrogen at positions 2 and 3 in the propionate chain were studied as a function of reaction time and extent of exchange of the 4'-hydrogen. No transfer of hydrogen from the 4' position to the side chain was observed, thereby eliminating mechanisms requiring hydride transfer via NADH between these positions. Catalytic rates in 1H2O vs. 2H2O revealed a 3-fold difference which was ascribed to a rate-limiting proton addition step. Similarly, a 5-fold decrease in Vmax was found for the reverse reaction when oxoimidazole[2,3-2H2]propionate was compared to unlabeled oxoimidazolepropionate. These data support a mechanism involving water addition across the conjugated double bond system of urocanate, rather than an internal oxidation--reduction process, yet NAD+ is required. A mechanism is proposed which uses electron delocalization in the imidazole nucleus, via an imidazole--NAD adduct, to facilitate water attack and subsequent formation of oxoimidazolepropionate.

Deuterium↗
PubMed1981

Cyclic adenosine 3',5'-monophosphate levels in Pseudomonas putida and Pseudomonas aeruginosa during induction and carbon catabolite repression of histidase synthesis.

Inducibility of histidase (histidine ammonia-lyase, EC 4.3.1.3) in Pseudomonas putida and Pseudomonas aeruginosa was observed to be strongly affected by succinate-provoked catabolite repression, but this did not occur as a consequence of reduced intracellular cyclic adenosine 3',5'-monophosphate levels, and repression could not be alleviated by exogenously added cyclic adenosine 3,'5'-monophosphate. Milder repression of histidase by lactate was also not reversed by the addition of cyclic adenosine 3',5'-monophosphate. These results, along with data showing intracellular cyclic adenosine 3',5'-monophosphate levels remained essentially constant during growth on such diverse carbon sources as histidine, acetamide, glucose, and succinate, indicated that catabolite repression of histidase synthesis by efficient carbon sources was not mediated through variations in internal cyclic adenosine 3,'5'-monophosphate.

Ammonia-Lyases↗
PubMed1980

Substrate-mediated inactivation of urocanase from Pseudomonas putida. Evidence for an essential sulfhydryl group.

Incubation of urocanase from Pseudomonas putida with either its substrate, urocanic acid, or product, 4'(5')-imidazolone-5'(4')-propionic acid, resulted in an oxygen-dependent inhibition of enzyme activity. Coincident with the inactivation was the stoichiometric incorporation of radioactivity from [14C]urocanate into the protein. NAD+ which is required for activity or urocanase was not directly involved in the inactivation process. The inactivation of urocanase was irreversible, could be partially blocked by the competitive inhibitor imidazolepropionate, and involved the modification of a single active-site thiol. The inhibition resulted from oxidative decomposition of 4'(5')-imidazolone-5'(4')-propionate but was not due to the formation of the major degradative product, 4-ketoglutaramate, since this compound was not an irreversible inactivator of urocanase although it did produce some inhibition at high concentrations. A mechanism is presented in which a reactive imine intermediate in the decomposition scheme is subject to nucleophilic attack by an active-site thiol, thereby generating a covalent enzyme--thioaminal adduct. These results emphasize the importance of a catalytic center sulfhydryl group for urocanase activity.

Aerobiosis↗
PubMed1978

Control of biodegradative threonine dehydratase inducibility by cyclic AMP in energy-restricted Escherichia coli.

To explain the requirement for anaerobic conditions in the induction of biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, measurements of cyclic AMP (cAMP) were made during aerobic and anaerobic growth and upon an aerobic-to-anaerobic transition. Internal cAMP levels were similar (5 to 10 muM) throughout exponential growth, whether aerobic or anaerobic, but only during anaerobiosis was threonine dehydratase synthesized. When an exponentially growing aerobic culture was made anaerobic, a sharp increase in internal cAMP was noted, reaching 300 muM within 10 min and declining thereafter to normal anaerobic levels. Threonine dehydratase synthesis was detected immediately after the attainment of peak cAMP levels and continued for several generations. A similar pattern but with less accumulation of cAMP and less threonine dehydratase production was also noted upon treatment of an aerobically growing culture with KCN. Pyruvate addition at the time of anaerobic shock severely affected both cAMP accumulation and threonine dehydratase synthesis; however, externally added cAMP could partially counter the pyruvate effect on enzyme synthesis. The conclusion was reached that conditions which resulted in a temporary energy deficit brought about the major accumulation of cAMP, and this elevated level served as a signal for initiation of threonine dehydratase synthesis to supply energy by the nonoxidative degradation of threonine.

Aerobiosis↗