The isolation and nucleotide sequence of the complex<i>AROM</i>locus of<i>Aspergillus nidulans</i>

Charles, Ian G.; Keyte, John W.; Brammar, William J.; Smith, Monica; Hawkins, Alastair R.

doi:10.1093/nar/14.5.2201

Cited by 128 publications

(92 citation statements)

References 39 publications

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“…All the alterations are distant from the active-site lysine residue, which the experiments described in this paper identify as residue 170. The sequences of the biosynthetic 3-dehydroquinase component of A. nidulans (Charles et al, 1985(Charles et al, , 1986 and S. cerevisiae (Duncan et al, 1987) arom polypeptides are also known, and these, together with the sequence of the N. crassa peptide CNIV/T determined in the present work, may be aligned with the revised E. coli sequence as shown in Fig. 6.…”

Section: Discussionmentioning

confidence: 95%

“…Limited-proteolysis experiments have shown that it is possible to isolate an arom fragment that carries both the 3-dehydroquinase and shikimate dehydrogenase activities, and is similar in size and structure to the bifunctional plant enzyme (Smith & Coggins, 1983;Coggins et al, 1985). The complete sequences of two fungal biosynthetic 3-dehydroquinases, the enzymes from Aspergillus nidulans (Charles et al, 1985(Charles et al, , 1986 and Saccharomyces cerevisiae (Duncan et al, 1987), are known.…”

Section: Introductionmentioning

confidence: 99%

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Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

Chaudhuri

Duncan

Graham

et al. 1991

Biochemical Journal

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The lysine residues involved in Schiff-base formation at the active sites of both the 3-dehydroquinase component of the pentafunctional arom enzyme of Neurospora crassa and of the monofunctional 3-dehydroquinase of Escherichia coli were labelled by treatment with 3-dehydroquinate in the presence of NaB3H4. Radioactive peptides were isolated by h.p.l.c. following digestion with CNBr (and in one case after further digestion with trypsin). The sequence established for the N. crassa peptide was ALQHGDVVKLVVGAR, and that for the E. coli peptide was QSFDADIPKIA. An amended nucleotide sequence for the E. coli gene (aroD) that encode 3-dehydroquinase is also presented, along with a revised alignment of the deduced amino acid sequences for the biosynthetic enzymes.

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Section: Discussionmentioning

confidence: 95%

Section: Introductionmentioning

confidence: 99%

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

Chaudhuri

Duncan

Graham

et al. 1991

Biochemical Journal

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“…Restriction fragments of interest were cloned into phages M13mpl8 and M13mpl9 (15) and were sequenced by the dideoxy nucleotide chain-termination method and 35S-substituted adenosine 5'-[thioltriphosphate (16). Gaps in the sequence were filled in by using oligonucleotides as specific sequencing primers (17). For "compressions" in DNA sequence, 7-deaza-dGTP was used in all four sequencing reaction mixes (18).…”

Section: Methodsmentioning

confidence: 99%

Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis.

Charles

Dougan

Pickard

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

150

116

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Protein P.69 is localized on the outer membrane of Bordetella pertussis and is one of the virulence factors believed to contribute to the disease state of whooping cough. We demonstrate that protein synthesis of P.69 is under genetic control of the vir locus. Using oligonucleotide probes derived from the protein sequence of a cyanogen bromide fragment, we have cloned the gene for P.69 from B. pertussis CN2992. Analysis of the DNA sequence reveals a G+C-rich gene capable of encoding a protein of 910 amino acids with a Mr of 93,478, suggesting that P.69 is a processed form of a larger precursor. In common with some of the genes in the pertussis toxin operon, the sequence CCTGG was found 5' to the ATG initiation codon. At the 3' end, 29 bases after the TAA stop codon, the sequence GTTTTTCCT was found and may have some function in transcription termination. A full-length clone ofthe gene for P.69 carried by the cosmid pBPI69 was unable to direct the expression of P.69 protein in an Escherichia coli host. The generation of P.69-fusion products allowed the detection of P.69-specific protein products synthesized in E. coli.Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are closely related pathogenic organisms. In humans, whooping cough is caused by B. pertussis or B. parapertussis. B. bronchiseptica is an animal pathogen, but this organism has also been isolated from children with whooping cough-like disease (1). There are many similarities between the diseases caused by these three species. All species undergo phenotypic changes that are genetically modulated by the vir locus, which regulates the expression of many proteins, some of which are apparently correlated with bacterial virulence and immunogenicity (2). These include filamentous hemagglutinin, adenylate cyclase (AdeCase), hemolysin, pili, and in B. pertussis, pertussis toxin (PTX

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“…This repressive effect is due to carbon catabolite repression rather than substrate exclusion [4]. The aromA gene encoding the pentafunctional AROM protein apparently evolved by multiple gene fusions to form a gene with a single open reading frame [5,6].…”

Section: Introductionmentioning

confidence: 99%

Control of metabolic flux through the quinate pathway in Aspergillus nidulans

Wheeler

Lamb

Hawkins

1996

Biochemical Journal

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The quinic acid utilization (qut) pathway in Aspergillus nidulans is a dispensable carbon utilization pathway that catabolizes quinate to protocatechuate via dehydroquinate and dehydroshikimate (DHS). At the usual in itro growth pH of 6.5, quinate enters the mycelium by means of a specific permease and is converted into PCA by the sequential action of the enzymes quinate dehydrogenase, 3-dehydroquinase and DHS dehydratase. The extent of control on metabolic flux exerted by the permease and the three pathway enzymes was investigated by applying the techniques of Metabolic Control Analysis. The flux control coefficients for each of the three quinate pathway enzymes were determined empirically, and the flux control coefficient of the quinate permease was inferred by use of the summation theorem. These measurements implied that, under the standard growth conditions used, the values for the flux control coefficients of the components of the quinate pathway were : quinate permease, 0.43 ; quinate dehydrogenase, 0.36 ; dehydroquinase, 0.18 ; DHS dehydratase, 0.03. Attempts to partially decouple quinate permease from the control over flux by measuring flux at pH 3.5 (when a significant percentage of the soluble quinate is protonated and able to enter the mycelium without the aid of a permease) led to an increase of approx. 50 % in the flux control coefficient for dehydroquinase. Taken together with the fact that A. nidulans has a very efficient pH homoeostasis mechanism, these experiments are consistent with the view that quinate permease exerts a high degree of control over pathway flux under the standard laboratory growth conditions at pH 6.5. The enzymes quinate dehydrogenase and 3-dehydroquinase have previously been overproduced in Escherichia coli, and protocols for their purification published. The remaining qut pathway enzyme DHS dehydratase was overproduced in E. coli and a purification protocol established. The purified DHS dehydratase was shown to have a K m of 530 µM for its substrate DHS and a

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The isolation and nucleotide sequence of the complexAROMlocus ofAspergillus nidulans

Cited by 128 publications

References 39 publications

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

Identification of the active-site lysine residues of two biosynthetic 3-dehydroquinases

Molecular cloning and characterization of protective outer membrane protein P.69 from Bordetella pertussis.

Control of metabolic flux through the quinate pathway in Aspergillus nidulans

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