The crystal structure of GMP synthetase serves as a prototype for two families of metabolic enzymes. The Class I glutamine amidotransferase domain of GMP synthetase is found in related enzymes of the purine, pyrimidine, tryptophan, arginine, histidine and folic acid biosynthetic pathways. This domain includes a conserved Cys-His-Glu triad and is representative of a new family of enzymes that use a catalytic triad for enzymatic hydrolysis. The structure and conserved sequence fingerprint of the nucleotide-binding site in a second domain of GMP synthetase are common to a family of ATP pyrophosphatases, including NAD synthetase, asparagine synthetase and argininosuccinate synthetase.

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.

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A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric Escherichia coli imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however, k cat /K m for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.

SummaryIn Pseudomonas putida, benzoate and 3-chlorobenzoate are converted to catechol and 3-chlorocatechol, respectively, which are then catabolized to tricarboxylic acid cycle intermediates via the catBCA and clcABD pathways. The catBCA and clcABD operons are regulated by homologous transcriptional activators CatR and ClcR. Previous studies have demonstrated that in addition to sequence similarities, CatR and ClcR share functional similarities which allow catR to complement clcR. In this study, we demonstrate that CatR activates the clcABD promoter in vitro without inducer, but more transcript is produced when inducer is added. DNase I footprinting and DNAbending analyses demonstrate that CatR binds to and bends the clcABD promoter to the same angle as does ClcR plus its inducer, 2-chloromuconate. This implies that CatR binds to the clc promoter in its active conformation. Transcription of the clcABD promoter by the ␣-subunit truncation mutant (␣-235) of RNA polymerase was sharply reduced, indicating that the ␣-subunit C-terminal domain is important. However, a small amount of transcript was produced under these conditions, indicating that other contact sites on the RNA polymerase may play a role in activation.

Ralstonia eutropha (formerly Alcaligenes eutrophus) NH9 degrades 3-chlorobenzoate via the modifiedortho-cleavage pathway. A ca. 5.7-kb six-gene cluster is responsible for chlorocatechol degradation: the cbnABCDoperon encoding the degradative enzymes (including orfX of unknown function) and the divergently transcribed cbnR gene encoding the LysR-type transcriptional regulator of the cbnoperon. The cbnRAB orfXCD gene cluster is nearly identical to the chlorocatechol genes (tcbRCD orfXEF) of the 1,2,4-trichlorobenzene-degrading bacterium Pseudomonas sp. strain P51. Transcriptional fusion studies demonstrated thatcbnR regulates the expression of cbnABCDpositively in the presence of either 3-chlorobenzoate or benzoate, which are catabolized via 3-chlorocatechol and catechol, respectively. In vitro transcription assays confirmed that 2-chloro-cis,cis-muconate (2-CM) andcis,cis-muconate (CCM), intermediate products from 3-chlorocatechol and catechol, respectively, were inducers of this operon. This inducer-recognizing specificity is different from those of the homologous catechol (catBCA) and chlorocatechol (clcABD) operons of Pseudomonas putida, in which only the intermediates of the regulated pathway, CCM forcatBCA and 2-CM for clcABD, act as significant inducers. Specific binding of CbnR protein to the cbnApromoter region was demonstrated by gel shift and DNase I footprinting analysis. In the absence of inducer, a region of ca. 60 bp from position −20 to position −80 upstream of the cbnAtranscriptional start point was protected from DNase I cleavage by CbnR, with a region of hypersensitivity to DNase I cleavage clustered at position −50. Circular permutation gel shift assays demonstrated that CbnR bent the cbnA promoter region to an angle of 78° and that this angle was relaxed to 54° upon the addition of inducer. While a similar relaxation of bending angles upon the addition of inducer molecules observed with the catBCA andclcABD promoters may indicate a conserved transcriptional activation mechanism of ortho-cleavage pathway genes, CbnR is unique in having a different specificity of inducer recognition and the extended footprint as opposed to the restricted footprint of CatR without CCM.

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.

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