Biomedical subjects

A T Phillips

Publications and source records attributed to A T Phillips.

At least 19 recordsLinked to original sources

PubMed2000

A method for parameter optimization in computational biology.

Models in computational biology, such as those used in binding, docking, and folding, are often empirical and have adjustable parameters. Because few of these models are yet fully predictive, the problem may be nonoptimal choices of parameters. We describe an algorithm called ENPOP (energy function parameter optimization) that improves-and sometimes optimizes-the parameters for any given model and for any given search strategy that identifies the stable state of that model. ENPOP iteratively adjusts the parameters simultaneously to move the model global minimum energy conformation for each of m different molecules as close as possible to the true native conformations, based on some appropriate measure of structural error. A proof of principle is given for two very different test problems. The first involves three different two-dimensional model protein molecules having 12 to 37 monomers and four parameters in common. The parameters converge to the values used to design the model native structures. The second problem involves nine bumpy landscapes, each having between 4 and 12 degrees of freedom. For the three adjustable parameters, the globally optimal values are known in advance. ENPOP converges quickly to the correct parameter set.

Algorithms↗

PubMed1998

Crystallization and preliminary X-ray studies of Pseudomonas putida histidine ammonium-lyase.

Histidine ammonium-lyase from P. putida was expressed in Escherichia coli, purified to homogeneity, and crystallized by the vapour-diffusion method using polyethylene glycol 3350 as the precipitant. The crystals, which diffract to at least 2.5 A resolution, exhibit the symmetry of space group P212121, with unit-cell parameters a = 89.7, b = 138.2 and c = 164.8 A. The asymmetric unit contains a tetramer, and the crystals have a Vm value of 2.41 A3 Da-1.

Bacterial Proteins↗

PubMed1997

Protein structure and energy landscape dependence on sequence using a continuous energy function.

We have recently described a new conformational search strategy for protein folding algorithms called the CGU (convex global underestimator) method. Here we use a simplified protein chain representation and a differentiable form of the Sun/Thomas/Dill energy function to test the CGU method. Standard search methods, such as Monte Carlo and molecular dynamics are slowed by kinetic traps. That is, the computer time depends more strongly on the shape of the energy landscape (dictated by the amino acid sequence) than on the number of degrees of freedom (dictated by the chain length). The CGU method is not subject to this limitation, since it explores the underside of the energy landscape, not the top. We find that the CGU computer time is largely independent of the monomer sequence for different chain folds and scales as O(n4) with chain length. By using different starting points, we show that the method appears to find global minima. Since we can currently find stable states of 36-residue chains in 2.4 hours, the method may be practical for small proteins.

Algorithms↗

PubMed1994

Ser-143 is an essential active site residue in histidine ammonia-lyase of Pseudomonas putida.

Site directed mutagenesis was used to investigate the role of Ser-143 in enzyme activity and as a point for attack by cyanide or L-cysteine, two irreversible inhibitors of histidine ammonia-lyase (histidase). Two mutant proteins, a S143A substitution and an A142S-S143A transposition, were made. Both mutant histidases completely lost all enzymatic activity. Western blots with anti-histidase antibodies revealed that the mutant proteins were being expressed at a level equal to that of the wild-type protein. The purified mutant proteins could not incorporate [14C]cyanide nor could they generate the UV-absorbing species normally observed when L-cysteine modifies wild-type histidase. These results support the hypothesis that Ser-143 is the binding site for an as yet unidentified histidase cofactor.

Amino Acid Sequence↗

PubMed1994

A revised map location for the histidine utilization genes in Pseudomonas putida.

The histidine utilization genes hutH and hutU of Pseudomonas putida ATCC 12633 have been mapped by interrupted mating and transduction to a location at approximately 43 minutes on the chromosome, closely linked to ser-800 and met-400 markers previously shown to be at 46 and 42 minutes, respectively. Since restriction enzyme mapping and cloning results have established that all genes associated with the hut pathway are contiguous, earlier maps of this strain which place these genes near 10 minutes on the chromosome in a superoperonic catabolic cluster are in error.

Chromosome Mapping↗

PubMed1994

1-amino-2-imidazol-4'-ylethylphosphonic acid is a potent reversible inhibitor of Pseudomonas putida histidine ammonia-lyase.

A phosphonic acid analogue of L-histidine, 1-amino-2-imidazol-4'-ylethylphosphonic acid (HisP), was identified as a reversible competitive inhibitor of histidine ammonia-lyase (histidase). The affinity of histidase for HisP was pH dependent, with Ki values of 0.28 microM and 10.4 microM compared to substrate Km values of 1 and 5 mM at pH 7 and 9, respectively. HisP did not appear to be a substrate for histidase. A twenty-fold molar excess of HisP over enzyme completely protected the active site of histidase from inactivation by 10 mM bisulfite. Neither isohistidine nor two other phosphonic acid compounds were inhibitory towards histidase when tested at 1 mM concentration.

Binding, Competitive↗

PubMed1993

Amino acid N-malonyltransferases from mung beans. Action on 1-aminocyclopropane-1-carboxylic acid and D-phenylalanine.

1-Aminocyclopropane-1-carboxylate (ACC) N-malonyltransferase from etiolated mung bean hypocotyls was examined for its relationship to D-phenylalanine N-malonyltransferase and other enzymes which transfer malonyl groups from malonyl-CoA to D-amino acids. Throughout a 3600-fold purification the ratio of D-phenylalanine N-malonyltransferase activity to ACC N-malonyltransferase activity was unchanged. Antibodies raised against purified ACC N-malonyltransferase 55-kDa protein were also able to precipitate all D-phenylalanine-directed activity from partially purified mung bean extracts. The irreversible inhibitors phenylglyoxal and tetranitromethane reduced malonyltransferase activity towards D-phenylalanine to the same extent as that for ACC. In addition, several other D-amino acids, particularly D-tryptophan and D-tyrosine, were able to inhibit action towards both ACC and D-phenylalanine. These lines of evidence suggest that a single enzyme is capable of promoting malonylation of both ACC and D-phenylalanine. Km values for D-phenylalanine and malonyl-CoA were found to be 48 and 43 microM, respectively; these values are 10-fold lower than the corresponding values when ACC was substrate. Coenzyme A was a noncompetitive (mixed type) product inhibitor towards malonyl-CoA at both unsaturated and saturated ACC concentrations. The enzyme was also inhibited uncompetitively at high concentrations of malonyl-CoA. We propose that the enzyme follows an Ordered Bi-Bi reaction pathway, with the amino acid substrate being bound initially.

Acyltransferases↗

PubMed1993

Identification of Ser143 as the site of modification in the active site of histidine ammonia-lyase.

Histidine ammonia-lyase (histidase) from Pseudomonas putida was irreversibly inactivated by L-cysteine at pH 10.5 in the presence of oxygen. Inactivation was accompanied by the formation of a new uv-absorbing species centered around 340 nm. L-[35S]cysteine labeling experiments revealed that 4 mol of L-cysteine was bound per mole of enzyme tetramer upon complete modification. However, the radiolabel was dissociated from the protein under denaturing conditions without loss of the 340-nm absorbance. Prior inactivation of histidase by cyanide, borohydride, or bisulfite precluded the formation of the 340-nm species in subsequent L-cysteine modification experiments. This suggests a common target site for modification of histidase by all of these reagents. Based on its strong absorbance at 340 nm an octapeptide was isolated from L-cysteine-inactivated histidase following trypsin and staphylococcal V8 protease digestion. Electrospray MS/MS revealed that this peptide (Gly138-SerValGlyAlaSerGlyAsp145) contained an unidentified modification of mass 184 Da located on Ser143. This peptide and the serine residue are conserved in all histidases and phenylalanine ammonia-lyases for which the amino acid sequence is available. Ser143 represents the binding site for an electrophilic cofactor required for histidase activity.

Amino Acid Sequence↗

PubMed1993

Identification of two new members of the 1-aminocyclopropane-1-carboxylate synthase-encoding multigene family in mung bean.

The key enzyme regulating ethylene biosynthesis in higher plants is 1-aminocyclopropane-1-carboxylate (ACC) synthase. In mung bean (MB), the existence of three genes encoding this enzyme has previously been reported [Botella et al., Plant Mol. Biol. 18 (1992) 793-797], one of which corresponds to a full-length indole-3-acetic acid-inducible cDNA [Botella et al., Plant Mol. Biol. (1992) 425-436]. In this paper we report the cloning of two new genomic sequences coding for ACC synthase in MB (MAC-4 and MAC-5). MAC-4 is 1340 bp long and encodes 388 amino acids (aa) while MAC-5 is 1393 bp long and encodes for 391 aa. Genomic Southern analysis suggests the existence of only one copy of each gene in the genome.

Amino Acid Sequence↗

PubMed1993

Purification and characterization of Pseudomonas putida histidine ammonia-lyase expressed in Escherichia coli.

Histidine ammonia-lyase (HAL) from Pseudomonas putida PRS1 contains a catalytically important electrophilic center reported to be dehydroalanine. Little is known about the origin of this group or its linkage to the protein. To initiate structural studies on this enzyme, P. putida HAL was purified from an Escherichia coli high-expression clone in which the HAL gene (hutH) was under the control of the lambda PL promoter on a plasmid vector. In this clone from 6 to 10% of the soluble cell protein after heat induction was HAL and approximately 200 mg of 95% pure HAL could be obtained from 120 g wet weight of cells in a 40 to 60% yield. The overexpressed protein was identical to P. putida HAL in native molecular weight (220 kDa), subunit composition (four identical subunits of 53 kDa each), affinity for substrate (L-histidine Km of 5.3 mM at pH 9.0), and its sensitivity to inactivation by cyanide and bisulfite. The N-terminal amino acid sequence was in agreement with the DNA-predicted sequence, indicating proper translational initiation. These features make this enzyme an appropriate candidate for protein structure investigations regarding the nature of the electrophilic center and its association with the protein.

Amino Acid Sequence↗

PubMed1992

Structural comparison of apomyoglobin and metaquomyoglobin: pH titration of histidines by NMR spectroscopy.

Proton NMR spectroscopy was applied to myoglobin in the ferric, water-liganded form (metMbH2O) and the apo form (apoMb) to probe the structure and stability of the latter. Proteins from sperm whale and horse skeletal muscles were studied to simplify the spectral assignment task. Nuclear Overhauser effects and the response of chemical shifts to variations of pH were used as indicators of residual native holoprotein structure in the apoprotein. The investigation was focused in the histidine side chains and their environment. In metMbH2O, the resonances of all imidazole rings not interacting with the heme were assigned by applying standard two-dimensional methods. These assignments were found to differ from those reported elsewhere [Carver, J. A., & Bradbury, J. H. (1984) Biochemistry 23, 4890-4905] except for His-12, -113, and -116. Only one histidine (His-36) has a pK(a) higher than 7, two (His-48 and His-113) have a pK(a) lower than 5.5, and two (His-24 and His-82) appear not to titrate between pH 5.5 and pH 10. In the apoproteins, the signals of His-113 and His-116, as well as those of His-24, -36, -48, and -119 previously assigned in the horse globin [Cocco, M. J.. & Lecomte, J. T. J. (1990) Biochemistry 29, 11067-11072], could be followed between pH 5 and pH 10. A comparison to the holoprotein data indicated that heme removal has limited effect on the pK(a) and the surroundings of these residues. Five additional histidines which occur in the two helices and connecting loops forming the heme binding site were identified in the horse apoprotein. Four of these were found to have pK(a) values lower than that expected of an exposed residue. The NOE and titration data were proposed to reflect the fact that several holoprotein structural elements, in particular outside the heme binding site, are maintained in the apoprotein. In the heme binding region of the apoprotein structure, the low pK(a)'s suggest local environments which are resistant to protonation.

Amino Acid Sequence↗

PubMed1992

Identification and characterization of three putative genes for 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments.

The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean.

Amino Acid Sequence↗

PubMed1992

Identification and characterization of a full-length cDNA encoding for an auxin-induced 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments and expression of its mRNA in response to indole-3-acetic acid.

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) is the key regulatory enzyme in the ethylene biosynthetic pathway. The identification and characterization of a full-length cDNA (pAIM-1) 1941 bp in length for indole-3-acetic acid (IAA)-induced ACC synthase is described in this paper. The pAIM-1 clone has an 87 bp leader and a 402 bp trailing sequence. The open reading frame is 1452 bp long encoding for a 54.6 kDa polypeptide (484 amino acids) which has a calculated isoelectric point of 6.0. In vitro transcription and translation experiments support the calculated molecular weight and show that the enzyme does not undergo processing. Eleven of the twelve amino acid residues which are conserved in aminotransferases are found in pAIM-1. The sequence for pMAC-1 which is one of the 5 genes we have identified in mung bean is contained in pAIM-1. pAIM-1 shares between 52 to 65% homology with previously reported sequences for ACC synthase at the protein level. There is little detectable pAIM-1 message found in untreated mung bean tissues; however, expression is apparent within 30 min following the addition of 10 microM IAA reaching a peak after approximately 5 h with a slight decrease in message after 12 h. These changes in message correlate with changes in ACC levels found in these tissues following treatment with 10 microM IAA.

Amino Acid Sequence↗

PubMed1990

Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida.

The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli. The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approximate molecular weight 53,600, in the location previously identified for the histidase gene by Tn1000 mutagenesis. Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence. This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits. Comparison of the P. putida histidase gene sequence with that of a Bacillus subtilis region encoding histidase revealed 42% identity at the protein level. Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.

Amino Acid Sequence↗

PubMed1990

Nucleotide sequence of the gene encoding the repressor for the histidine utilization genes of Pseudomonas putida.

The hutC gene of Pseudomonas putida encodes a repressor which, in combination with the inducer urocanate, regulates expression of the five structural genes necessary for conversion of histidine to glutamate, ammonia, and formate. The nucleotide sequence of the hutC region was determined and found to contain two open reading frames which overlapped by one nucleotide. The first open reading frame (ORF1) appeared to encode a 27,648-dalton protein of 248 amino acids whose sequence strongly resembled that of the hut repressor of Klebsiella aerogenes (A. Schwacha and R. A. Bender, J. Bacteriol. 172:5477-5481, 1990) and contained a helix-turn-helix motif that could be involved in operator binding. The gene was preceded by a sequence which was nearly identical to that of the operator site located upstream of hutU which controls transcription of the hutUHIG genes. The operator near hutC would presumably allow the hut repressor to regulate its own synthesis as well as the expression of the divergent hutF gene. A second open reading frame (ORF2) would encode a 21,155-dalton protein, but because this region could be deleted with only a slight effect on repressor activity, it is not likely to be involved in repressor function or structure.

Amino Acid Sequence↗

PubMed1989

Identification of multiple repressor recognition sites in the hut system of Pseudomonas putida.

The hutC gene in Pseudomonas putida encodes a repressor protein that negatively regulates the expression of all hut genes. We have overexpressed this cloned hutC gene in Escherichia coli to identify P. putida hut regions that could specifically bind the repressor. Ten restriction fragments, some of which were partially overlapping and spanned the coding portions of the P. putida hut region, were labeled and tested for their ability to recognize repressor in a filter binding assay. This procedure identified three binding sites, thus supporting previous indications that there were multiple operons. A 1.0-kilobase-pair SalI restriction fragment contained the operator region for the hutUHIG operon, whereas a 1.9-kilobase-pair SmaI fragment contained the hutF operator. A 2.9-kilobase-pair XhoI segment appeared to contain the third operator, corresponding to a separate and perhaps little used control region for hutG expression only. The addition of urocanate, the normal inducer, caused dissociation of all operator-repressor complexes, whereas N-formylglutamate, capable of specifically inducing expression of the hutG gene, inhibited binding only of repressor to fragments containing that gene. Formylglutamate did not affect the action of urocanate on the repressor-hutUHIG operator complex, indicating that it binds to a site separate from urocanate on the repressor. DNA footprinting and gel retardation analyses were used to locate more precisely the operator for the hutUHIG operon. A roughly 40-base-pair portion was identified which contained a 16-base-pair region of dyad symmetry located near the transcription initiation site for this operon.

Base Sequence↗

PubMed1988

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyls.

1-Aminocyclopropane-1-carboxylate (ACC) synthase, EC 4.4.1.14, was purified to homogeneity from etiolated mung bean hypocotyl segments. This was made possible by the ability to elevate the enzyme level markedly through hormone treatments and by stabilization of the enzyme with high phosphate concentrations. The four-step procedure resulted in 1050-fold purification with 25% yield, and consisted of stepwise elution from hydroxylapatite, chromatography on phenyl-Sepharose CL-4B, gradient elution from hydroxylapatite, and fast protein liquid chromatography (FPLC) on a MonoQ anion-exchange column. FPLC-purified ACC synthase migrated as a single band of Mr 65,000 on denaturing polyacrylamide gel electrophoresis. The molecular weight of native enzyme by Bio-Gel A-0.5 M chromatography was 125,000, indicating that the enzyme probably exists as a dimer of identical 65,000 Mr subunits. The mung bean ACC synthase exhibited a pH optimum of 8.0 for activity and a Km for S-adenosylmethionine (AdoMet) of 55 microM at 30 degrees C. It exhibited an Arrhenius activation energy of 12 kcal mol-1 degree-1 and was inactivated at temperatures in excess of 40 degrees C. The specific activity for pure ACC synthase was 21 mumol of ACC formed/mg protein/h when determined under optimal conditions with 400 microM AdoMet.

Adenine↗

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↗