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PubMed1969

In vitro formation of an active multienzyme complex in the tryptophan pathway of Neurospora crassa.

A multienzyme complex that catalyzes the anthranilate synthetase, phosphoribosylanthranilate (PRA) isomerase, and indoleglycerolphosphate (InGP) synthetase reactions was produced in vitro when extracts from a tryp-1 mutant and a tryp-2 mutant of Neurospora crassa were mixed. The sedimentation values and the molecular weights for the interacting components obtained from the mutants were estimated by sucrose gradients and by gel filtration on Sephadex columns. The component coded for by the tryp-2 gene (a-component) which is present in the tryp-1-17 mutant has a sedimentation coefficient of 4.5S and molecular weight of 70,000. The component coded for by the tryp-1 gene (i-component) which is present in the tryp-2-6 mutant has a sedimentation coefficient 7.5S and a molecular weight of 170,000. The complex formed in vitro had similar properties to those previously reported for the wild-type complex. A Q(10) value of 3 and an apparent activation energy of 18,000 cal/mole were calculated for the formation of the complex from the two components.

Centrifugation, Zonal↗
PubMed1989

Identification and regulation of a rat liver cDNA encoding farnesyl pyrophosphate synthetase.

CR39 is a cholesterol-repressible rat liver cDNA previously isolated by differential hybridization (Clarke, C.F., Tanaka, R.D., Svenson, K., Wamsley, M., Fogelman, A.M., and Edwards, P.A. (1987) Mol. Cell. Biol. 7, 3138-3146). To precisely identify the function of CR39 a fusion protein was constructed that contained the amino-terminal region of the bacterial protein anthranilate synthetase fused to the full length CR39 polypeptide. Affinity purified antisera directed against the fusion protein inactivated rat liver cytosolic prenyltransferase activity in vitro. In addition, affinity purified antisera made to purified chicken prenyltransferase cross-reacted with the fusion protein containing CR39. Rat hepatic prenyltransferase activity and enzyme mass were quantitated in animals fed diets or drugs known to alter endogenous cholesterol biosynthesis. Rats fed a diet supplemented with cholestyramine and mevinolin showed a 3.5-fold increase in activity and a 5.0-fold increase in mass of cytosolic prenyltransferase. A diet supplemented with cholesterol resulted in approximately a 4.0-fold decrease in hepatic enzyme activity and a 10-fold decrease in enzyme mass. Under these same dietary regimens the mass of prenyltransferase in the testes remained unchanged. We conclude that CR39 encodes the prenyltransferase of cholesterol biosynthesis, farnesyl pyrophosphate synthetase. Furthermore, in the liver this enzyme shows coordinate regulation with two other enzymes, 3-hydroxy-3-methylglutaryl-CoA reductase and 3-hydroxy-3-methylglutaryl-CoA synthase, in response to cholesterol feeding and hypocholesterolemic drugs.

Amino Acid Sequence↗
PubMed1988

Chloroplast rpoA, rpoB, and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription.

The purpose of this study was to determine the relationship between putative chloroplast RNA polymerase subunit genes and known chloroplast transcriptional activities. We have prepared fusion polypeptide genes from fragments of chloroplast DNA homologous to bacterial RNA polymerase subunit genes and expression vectors carrying portions of the anthranilate synthetase gene (trpE). Fusion proteins for chloroplast homologs of the RNA polymerase alpha (rpoA), beta (rpoB), and beta' (rpoC) subunits were obtained from these genes. The fusion polypeptides synthesized by Escherichia coli in vivo were purified and used as antigens for production of rabbit polyclonal anti-RNA polymerase subunit-specific antibodies. The purified antibodies were able to immobilize chloroplast DNA-dependent RNA polymerases from spinach, pea, and Euglena gracilis. In addition, the soluble chloroplast RNA polymerase activity in tRNA and mRNA synthesis was strongly inhibited by these antibodies under conditions which had little effect on transcription by the chloroplast transcriptionally active chromosome that preferentially transcribed rRNA genes (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259: 14880-14887). From these data we conclude that the chloroplast genes homologous to bacterial RNA polymerase subunit genes are expressed in vivo and that the protein products specify at least three of the components of the chloroplast RNA polymerase(s) involved in tRNA and mRNA transcription.

Amino Acid Sequence↗
PubMed2001

Oligomerization of uniquely folded mini-protein motifs: development of a homotrimeric betabetaalpha peptide.

The discovery of a discretely folded homotrimeric betabetaalpha motif (BBAT1) was recently reported (J. Am. Chem. Soc. 2001, 123, 1002-1003). Herein the design, synthesis, and analysis of a small library of peptides which led to the isolation of BBAT1 is described. betabetaalpha peptides based on the monomeric sequence of BBA5 (Folding Des. 1998, 120, 95-103) were synthesized to include the anthranilic acid/nitrotyrosine fluorescence quenching pair to rapidly detect interpeptide association. In the first generation of peptides synthesized, truncations in the loop region connecting the beta-hairpin to the alpha-helix revealed that a two-residue deletion in the loop promoted an interpeptide association as detected by fluorescence quenching. An additional library of 22 loop-truncated betabetaalpha peptides was subsequently synthesized to include a variety of sequence mutations in an effort to enhance the observed peptide-peptide binding. From the fluorescence quenching screen, peptide B2 was found to possess the strongest fluorescence-quenching response, indicative of a strong peptide-peptide association. Due the poor solubility of peptide B2, the S-methylated cysteine at position 9 in the loop was substituted with a glycine to generate peptide BBAT1 which possessed greatly improved water solubility and formed discrete trimers. The successful design of this oligomeric betabetaalpha structure will likely aid the design of more complex alpha-beta superstructures and further our understanding of the factors controlling protein-protein interactions at alpha-beta protein interfaces.

Amino Acid Motifs↗
PubMed1990

An 8-fold beta alpha barrel protein with redundant folding possibilities.

Protein sequences containing redundant segments of secondary structure at both termini have the choice a priori of folding into several possible circularly permuted variants of the wild-type tertiary structure. To test this hypothesis the gene of phosphoribosyl anthranilate isomerase from yeast, which is a single-domain 8-fold beta alpha barrel protein, was modified to produce a 10-fold beta alpha homologue in Escherichia coli. It contained a duplicate of the two C-terminal beta alpha units of supersecondary structure fused to its N-terminus. Most of the protein was recovered from the insoluble fraction of disrupted cells by dissolution in guanidinium chloride solutions and refolding. Pristine protein was purified from the soluble fraction. The purified (beta alpha)10 proteins were enzymically almost fully active. Absorbance, fluorescence and circular dichroism spectra as well as the reversible unfolding behaviour of both proteins were also very similar to the properties of the original (beta alpha)8 protein. Digestion with endopeptidases converted both the pristine and the refolded (beta alpha)10 variant to the same large fragment that had the N-terminal sequence and mol. wt of the wild-type (beta alpha)8 protein. The data suggest that the folding of the (beta alpha)10 variant is controlled thermodynamically both in vivo and in vitro.

Aldose-Ketose Isomerases↗
PubMed1987

Three-dimensional structure of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli.

N-(5'-Phosphoribosyl)anthranilate isomerase-indole-3-glycerol-phosphate synthase from Escherichia coli is a monomeric bifunctional enzyme of Mr 49,500 that catalyzes two sequential reactions in the biosynthesis of tryptophan. The three-dimensional structure of the enzyme has been determined at 2.8-A resolution by x-ray crystallography. The two catalytic activities reside on distinct functional domains of similar folding, that of an eightfold parallel beta-barrel with alpha-helices on the outside connecting the beta-strands. Both active sites were located with an iodinated substrate analogue and found to be in depressions on the surface of the domains created by the outward-curving loops between the carboxyl termini of the beta-sheet strands and the subsequent alpha-helices. They do not face each other, making "channeling" of the substrate between active sites virtually impossible. Despite the structural similarity of the two domains, no significant sequence homology was found when topologically equivalent residues were compared.

Aldose-Ketose Isomerases↗
PubMed2003

Eukaryotic NAD+ synthetase Qns1 contains an essential, obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase.

NAD+ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADP-ribosyl)ation. NAD+ synthetase catalyzes the final step in NAD+ synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD+ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD+ synthetases are ammonia-dependent. Earlier, we discovered that glutamine-dependent NAD+ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD+ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD+ synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD+ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilase-related domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 A from one active site to another within Qns1 monomers.

Amide Synthases↗
PubMed2000

Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage.

Aromatic compound degradation in six bacteria representing an ecologically important marine taxon of the alpha-proteobacteria was investigated. Initial screens suggested that isolates in the Roseobacter lineage can degrade aromatic compounds via the beta-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Six Roseobacter isolates were screened for the presence of protocatechuate 3,4-dioxygenase, a key enzyme in the beta-ketoadipate pathway. All six isolates were capable of growth on at least three of the eight aromatic monomers presented (anthranilate, benzoate, p-hydroxybenzoate, salicylate, vanillate, ferulate, protocatechuate, and coumarate). Four of the Roseobacter group isolates had inducible protocatechuate 3, 4-dioxygenase activity in cell extracts when grown on p-hydroxybenzoate. The pcaGH genes encoding this ring cleavage enzyme were cloned and sequenced from two isolates, Sagittula stellata E-37 and isolate Y3F, and in both cases the genes could be expressed in Escherichia coli to yield dioxygenase activity. Additional genes involved in the protocatechuate branch of the beta-ketoadipate pathway (pcaC, pcaQ, and pobA) were found to cluster with pcaGH in these two isolates. Pairwise sequence analysis of the pca genes revealed greater similarity between the two Roseobacter group isolates than between genes from either Roseobacter strain and soil bacteria. A degenerate PCR primer set targeting a conserved region within PcaH successfully amplified a fragment of pcaH from two additional Roseobacter group isolates, and Southern hybridization indicated the presence of pcaH in the remaining two isolates. This evidence of protocatechuate 3, 4-dioxygenase and the beta-ketoadipate pathway was found in all six Roseobacter isolates, suggesting widespread abilities to degrade aromatic compounds in this marine lineage.

Adipates↗
PubMed2003

Lateral gene transfer and ancient paralogy of operons containing redundant copies of tryptophan-pathway genes in Xylella species and in heterocystous cyanobacteria.

BACKGROUND: Tryptophan-pathway genes that exist within an apparent operon-like organization were evaluated as examples of multi-genic genomic regions that contain phylogenetically incongruous genes and coexist with genes outside the operon that are congruous. A seven-gene cluster in Xylella fastidiosa includes genes encoding the two subunits of anthranilate synthase, an aryl-CoA synthetase, and trpR. A second gene block, present in the Anabaena/Nostoc lineage, but not in other cyanobacteria, contains a near-complete tryptophan operon nested within an apparent supraoperon containing other aromatic-pathway genes. RESULTS: The gene block in X. fastidiosa exhibits a sharply delineated low-GC content. This, as well as bias of codon usage and 3:1 dinucleotide analysis, strongly implicates lateral gene transfer (LGT). In contrast, parametric studies and protein tree phylogenies did not support the origination of the Anabaena/Nostoc gene block by LGT. CONCLUSIONS: Judging from the apparent minimal amelioration, the low-GC gene block in X. fastidiosa probably originated by LGT at a relatively recent time. The surprising inability to pinpoint a donor lineage still leaves room for alternative, albeit less likely, explanations other than LGT. On the other hand, the large Anabaena/Nostoc gene block does not seem to have arisen by LGT. We suggest that the contemporary Anabaena/Nostoc array of divergent paralogs represents an ancient ancestral state of paralog divergence, with extensive streamlining by gene loss occurring in the lineage of descent representing other (unicellular) cyanobacteria.

Amino Acid Sequence↗
PubMed1977

[Experience with a locally applicable antirheumatic (author's transl)].

Clinical trials with 2-(2-hydroxyethoxy)-ethyl-N-(a,a,a-trifluoro-m-tolyl)-anthranilate (etofenamate, active principle of Rheumon Gel) were carried out on a total of 69 patients suffering from extra-articular rheumatic complaints and painful spinal syndromes as well as from post-operative and post-accident complaints. Criteria for assessment included spontaneous pain, pressure pain and pain on movement and also limitation in joint mobility. The new gel formulation proved to have a good analgesic efficacy both in a double blind trial against placebo gel and the open study. Those patients treated with Rheumon Gel showed improvement in the symptoms spontaneous pain and pain on movement, in a large number of which patients showing improvement, in a large number of which patients showing improvement the spontaneous pain was completely eliminated. The differences in therapeutic effect compared to the placebo group as well as those of the final clinical assessment were statistically significant. Particularly good therapeutic results were observed in extra-articular rheumatic complaints and in activated arthroses. The skin tolerance of Rheumon Gel was found to be excellent. The gel form permits good and rapid penetration through the skin. The nursing staff also willingly administered the product because of its pleasant protective properties for skin and clothing.

Administration, Topical↗
PubMed2005

Crystal structure of vinorine synthase, the first representative of the BAHD superfamily.

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-angstrom resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.

Acetyltransferases↗
PubMed1997

Crystal structure at 2.0 A resolution of phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima: possible determinants of protein stability.

The structural basis of thermostability of proteins is of great scientific and biotechnological interest. Differences in the X-ray structues of orthologous proteins from hyperthermophilic and mesophilic organisms can indicate crucial stabilizing interactions. To this end the crystal structure of dimeric phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima (tPRAI) was determined using phases derived from the isomorphous replacement method and was refined at 2.0 A resolution. The comparison to the known 2.0 A structure of PRAI from Escherichia coli (ePRAI) shows that tPRAI has the complete TIM- or (beta alpha)8-barrel fold, whereas helix alpha5 in ePRAI is replaced by a loop. The subunits of tPRAI associate via the N-terminal faces of their central beta-barrels. Two long, symmetry-related loops that protrude reciprocally into cavities of the other subunit provide for multiple hydrophobic interactions. Moreover, the side chains of the N-terminal methionines and the C-terminal leucines of both subunits are immobilized in a hydrophobic cluster, and the number of salt bridges is increased in tPRAI. These features appear to be mainly responsible for the high thermostability of tPRAI. In contrast to other hyperthermostable enzymes, tPRAI at 25 degrees C is catalytically more efficient than ePRAI, mainly due to its small K(M) value for the substrate [Sterner, R., Kleemann, G. R., Szadkowski, H., Lustig, A., Hennig, M., & Kirschner, K. (1996) Protein Sci. 5, 2000-2008]. The increased number of hydrogen bonds between the phosphate ion and tPRAI compared to ePRAI could be responsible for this effect.

Aldose-Ketose Isomerases↗
PubMed2006

Substitutions of the "bridging" aspartate 178 result in profound changes in the reactivity of the Rieske center of phthalate dioxygenase.

Phthalate dioxygenase (PDO) and its reductase (PDR) are parts of a two-component Rieske oxygenase system that initiates the aerobic breakdown of phthalate by forming cis-4,5-dihydro-4,5-dihydroxyphthalate. Aspartate D178 in PDO, which lies between the Rieske [2Fe-2S] center of one subunit and the mononuclear center of the adjacent subunit, is highly conserved among the Rieske dioxygenases. The analogous aspartate has been implicated in electron transfer in naphthalene dioxygenase and in substrate binding and oxygen reactivity in anthranilate dioxygenase. Substitution of D178 with alanine or asparagine in PDO resulted in proteins with significantly increased Fe(II) dissociation constants. The rates of oxidation of the reduced Rieske centers in D178A and D178N were decreased by more than 10(4)-fold; only part of the loss of activity can be attributed to depletion of iron from the mononuclear centers. Reduction of PDO by reduced PDR was also slower in the D178A and D178N variants. Observed decreases in turnover rates of D178A and D178N compared to that of wild-type (WT) PDO (>10(2)-fold) can be ascribed to the cumulative effect of the low intrinsic iron content of the D178A and D178N mutants and the combination of the decreased rates of Rieske center reduction and oxidation. The coupling of dihydrodiol formation approached 100% in WT PDO but was only approximately 16% in D178A and approximately 7% in D178N. In single-turnover experiments, very small amounts of DHD were produced by D178A and D178N "as purified". The presence of saturating amounts of ferrous ion improved coupling to nearly 100% for the D178N variant but only slightly improved coupling for D178A. Thus, although hydroxylation is still possible in the variants, the reactions are largely uncoupled due to slow intramolecular electron transfer rates and the apparent weak binding of iron at the mononuclear centers.

Amino Acid Substitution↗
PubMed1995

Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa.

Salicylate is a precursor of pyochelin in Pseudomonas aeruginosa and both compounds display siderophore activity. To elucidate the salicylate biosynthetic pathway, we have cloned and sequenced a chromosomal region of P. aeruginosa PAO1 containing two adjacent genes, designated pchB and pchA, which are necessary for salicylate formation. The pchA gene encodes a protein of 52 kDa with extensive similarity to the chorismate-utilizing enzymes isochorismate synthase, anthranilate synthase (component I) and p-aminobenzoate synthase (component I), whereas the 11 kDa protein encoded by pchB does not show significant similarity with other proteins. The pchB stop codon overlaps the presumed pchA start codon. Expression of the pchA gene in P. aeruginosa appears to depend on the transcription and translation of the upstream pchB gene. The pchBA genes are the first salicylate biosynthetic genes to be reported. Salicylate formation was demonstrated in an Escherichia coli entC mutant lacking isochorismate synthase when this strain expressed both the pchBA genes, but not when it expressed pchB alone. By contrast, an entB mutant of E. coli blocked in the conversion of isochorismate to 2,3-dihydro-2,3-dihydroxybenzoate formed salicylate when transformed with a pchB expression construct. Salicylate formation could also be demonstrated in vitro when chorismate was incubated with a crude extract of P. aeruginosa containing overproduced PchA and PchB proteins; salicylate and pyruvate were formed in equimolar amounts. Furthermore, salicylate-forming activity could be detected in extracts from a P. aeruginosa pyoverdin-negative mutant when grown under iron limitation, but not with iron excess. Our results are consistent with a pathway leading from chorismate to isochorismate and then to salicylate plus pyruvate, catalyzed consecutively by the iron-repressible PchA and PchB proteins in P. aeruginosa.

Amino Acid Sequence↗
PubMed2006

Differentiation of carbazole catabolic operons by replacement of the regulated promoter via transposition of an insertion sequence.

The carbazole catabolic car operons from Pseudomonas resinovorans CA10 and Janthinobacterium sp. J3 have nearly identical nucleotide sequences in their structural and intergenic regions but not in their flanking regions. Transposition of ISPre1 from the anthranilate catabolic ant operon located an inducible promoter Pant upstream of the carCA10 operon, which is regulated by the AraC/XylS family activator AntR in response to anthranilate. The transposed Pant drives transcription of the carCA10 operon, which is composed of the car-AaAaBaBbCAcAdDFECA10 structural genes. Transcriptional fusion truncating Pant upstream of carAaCA10 resulted in constitutive luciferase expression. Primer extension analysis identified a transcription start point of the constitutive mRNA of the carCA10 operon at 385 nucleotides upstream of the carAaCA10 translation start point, and the PcarAa promoter was found. On the other hand, a GntR family regulatory gene carRJ3 is divergently located upstream of the carJ3 operon. The Pu13 promoter, required for inducible transcription of the carJ3 operon in the presence of carbazole, was identified in the region upstream of carAaJ3, which had been replaced with the Pant promoter in the carCA10 operon. Deletion of carRJ3 from a transcriptional fusion resulted in high level constitutive expression from Pu13. Purified CarRJ3 protein bound at two operator sequences OI and OII, showing that CarRJ3 directly represses Pu13 in the absence of its inducer, which was identified as 2-hydroxy-6-oxo-6-(2'-aminophenyl)hexa-2,4-dienoate, an intermediate of the carbazole degradation pathway.

Base Sequence↗
PubMed2005

Characterization of the sorption of an anthranilate fungicide in soil using thermal analytical and mineralogical techniques.

The sorption of pesticides to soil particles has implications for their distribution and fate in the soil environment. A batch equilibrium technique was used to investigate sorption of the fungicide AEC623892 to intact and hydrogen-peroxide-treated whole soils and several particle-size fractions isolated from them. Sorption of AEC623892 to the soil as a whole was low. K(f oc) values measured in the whole soils were 169.2 and 41.9 ml g(-1) for Soil A and Soil B respectively. The highest values of K(f oc) were measured in soil particle-size fractions <53 microm (266.5 ml g(-1) in the 2-20 microm fraction of Soil A; 471.9 ml g(-1) in the 20-53 microm fraction of Soil B). Sorption was most irreversible in the 2-20 microm fractions. Overall, treatment of soil particle-size fractions with hydrogen peroxide resulted in lower values of K(f oc) (112.3 ml g(-1) in Soil A whole soil and 30.9 ml g(-1) in Soil B whole soil). In both soils, the maximum sorption among hydrogen-peroxide-treated samples was observed in the <2 microm fraction (166.6 and 311.0 ml g(-1) for Soil A and Soil B, respectively). Investigation of the mineralogical composition of the soils suggested that the clay mineralogy (dominated by kaolinite and illite) is less likely to account for the differences in sorption observed than differences in the characteristics of the soil organic matter. Thermal analysis of the different soil fractions indicated that hydrogen peroxide treatment preferentially removed aliphatic fractions of organic matter, but had less effect on lignin-like, aromatic fractions.

Adsorption↗
PubMed2001

Cloning and expression of the benzoate dioxygenase genes from Rhodococcus sp. strain 19070.

The bopXYZ genes from the gram-positive bacterium Rhodococcus sp. strain 19070 encode a broad-substrate-specific benzoate dioxygenase. Expression of the BopXY terminal oxygenase enabled Escherichia coli to convert benzoate or anthranilate (2-aminobenzoate) to a nonaromatic cis-diol or catechol, respectively. This expression system also rapidly transformed m-toluate (3-methylbenzoate) to an unidentified product. In contrast, 2-chlorobenzoate was not a good substrate. The BopXYZ dioxygenase was homologous to the chromosomally encoded benzoate dioxygenase (BenABC) and the plasmid-encoded toluate dioxygenase (XylXYZ) of gram-negative acinetobacters and pseudomonads. Pulsed-field gel electrophoresis failed to identify any plasmid in Rhodococcus sp. strain 19070. Catechol 1,2- and 2,3-dioxygenase activity indicated that strain 19070 possesses both meta- and ortho-cleavage degradative pathways, which are associated in pseudomonads with the xyl and ben genes, respectively. Open reading frames downstream of bopXYZ, designated bopL and bopK, resembled genes encoding cis-diol dehydrogenases and benzoate transporters, respectively. The bop genes were in the same order as the chromosomal ben genes of P. putida PRS2000. The deduced sequences of BopXY were 50 to 60% identical to the corresponding proteins of benzoate and toluate dioxygenases. The reductase components of these latter dioxygenases, BenC and XylZ, are 201 residues shorter than the deduced BopZ sequence. As predicted from the sequence, expression of BopZ in E. coli yielded an approximately 60-kDa protein whose presence corresponded to increased cytochrome c reductase activity. While the N-terminal region of BopZ was approximately 50% identical in sequence to the entire BenC or XylZ reductases, the C terminus was unlike other known protein sequences.

Benzoates↗
PubMed1993

Imidazole glycerol phosphate synthase: the glutamine amidotransferase in histidine biosynthesis.

Two proteins essential for the biosynthesis of the amino acid histidine in Escherichia coli have been overexpressed and purified to apparent homogeneity. The protein encoded by the hisF gene has an ammonia-dependent activity that results in the conversion of the biosynthetic intermediate N1-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4- carboxamide ribonucleotide (PRFAR) to imidazole glycerol phosphate (IGP) and 5-aminoimidazole-4-carboxamido-1-beta-D- ribofuranosyl 5'-monophosphate (AICAR). The second protein encoded by the hisH gene exhibits no detectable catalytic properties with biosynthetic intermediate PRFAR, glutamine, or ammonia. In combination, the proteins are capable of a stoichiometric conversion of glutamine and PRFAR to form AICAR, IGP, and glutamate. Neither protein alone is capable of mediating a conversion of the nucleotide substrate to a free metabolic intermediate. The HisH and HisF proteins form a stable 1:1 dimeric complex that constitutes the IGP synthase holoenzyme. Steady-state kinetic parameters for the holoenzyme indicate that glutamine is a more efficient substrate relative to ammonium ion by a factor of 10(3). The HisF subunit will support an ammonia-dependent reaction with a turnover number similar to that of the holoenzyme with glutamine. The glutaminase activity for the holoenzyme is 0.8% of that in the presence of the nucleotide substrate PRFAR. There are critical subunit interactions that mediate the catalytic properties for glutamine hydrolysis. The catalytic turnover of glutamine can be increased up to 37-fold by the addition of either the product IGP or the biosynthetic precursor N1-[(5'-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (5'-ProFAR). The mechanistic significance of this glutaminase activity compared to other trpG type glutamine amidotransferases is discussed.

Anthranilate Synthase↗