p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA.

p-Aminobenzoic acid (PABA) is an important precursor in the bacterial biosynthetic pathway for folate enzymes. This biosynthesis requires three separate proteins: PabA, PabB, and PabC. Together PabA and PabB convert glutamine and chorismate to glutamate and 4-amino-4-deoxychorismate. This aminochorismate is subsequently transformed to PABA by PabC. In this study, PabA from Escherichia coli has been purified to homogeneity from an overproducing construct and found to have no detectable glutaminase activity until addition of the E. coli PabB subunit. PabB forms a 1:1 complex with PabA to yield a glutaminase k(cat) of 17 min-1. The addition of chorismate, the substrate of PabB, induces a 2-fold increase of k(cat) as well as a 3-fold increase of Km for glutamine. The PabA/PabB complex has Kd less than 10(-8) M but does not form a stable complex isolable by gel filtration. Studies with the glutamine affinity label diazooxonorleucine (DON) reveal it is an inactivator of the glutaminase activity of the PabA/PabB complex, but DON does not alkylate and inactivate PabA alone. Similarly, while isolated PabA shows no tendency to form a glutamyl-enzyme intermediate, the PabA/PabB complex forms a covalent intermediate with [14C]glutamine on PabA that accumulates to 0.56 mol/mol in hydrolytic turnover. PabA is thus a conditional glutaminase, activated by 1:1 complexation with PabB.

An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene.

McDonald and Burke (J. Bacteriol. 149:391-394, 1982) previously cloned a sulfanilamide-resistance gene, sul, residing on a 4.9-kb segment of Bacillus subtilis chromosomal DNA, into plasmid pUB110. In this study we determined the nucleotide sequence of the entire 4.9-kb fragment. Genes identified on the fragment include pab, trpG, pabC, sul, one complete unidentified open reading frame, and one incomplete unidentified open reading frame. The first three of these genes, pab, trpG, and pabC, are required for synthesis of p-aminobenzoic acid. The trpG gene encodes an amphibolic glutamine amidotransferase required for synthesis of both p-aminobenzoate and anthranilate, the latter an intermediate in the tryptophan biosynthetic pathway. The pabC gene may encode a B. subtilis analog of enzyme X, an enzyme needed for p-aminobenzoate synthesis in Escherichia coli. The sul gene probably encodes dihydropteroate synthase, the enzyme responsible for formation of 7,8-dihydropteroate, the immediate precursor of folic acid. All six of the cloned genes are arranged in a single operon. Since all four of the identified genes are needed for folate biosynthesis, we refer to this operon as a folic acid operon. Expression of the trpG gene is known to be negatively controlled by tryptophan. We propose that this regulation is at the level of translation. This hypothesis is supported by the finding of an apparent Mtr-binding site which overlaps with the trpG ribosome-binding site.

Auxotrophic Salmonella typhi as live vaccine.

Salmonella typhi 541Ty has deletions at aroA and purA, causing requirement for aromatic metabolites (including p-aminobenzoate) and for adenine. None of 36 volunteers who drank 10(8) to 10(10) bacteria of 541Ty or its Vi-negative mutant 543Ty showed any adverse effect; all gave evidence of cellular immune response but only a few had serum titre increases. S. typhimurium experiments (at the Wellcome Research Laboratories and at Stanford) show that adenine requirement may reduce both bacterial survival in mouse tissues and live-vaccine efficacy. S. typhi attenuated only by block(s) in aromatic biosynthesis may be more effective as oral-route live vaccine.

Mechanisms of sulfadoxine resistance in Plasmodium falciparum.

Three possible mechanisms of resistance to sulfadoxine were investigated in resistant Plasmodium falciparum: drug uptake, metabolism and alternate pathways. Uptake of [35S] sulfadoxine was markedly reduced in resistant plasmodia. By Thin Layer Radiochromatography it could be demonstrated that plasmodia do not metabolize sulfadoxine to pharmacologically inactive forms. Metabolism of sulfadoxine to the toxic analog of dihydropteroate is reduced in resistant plasmodia. Para-aminobenzoic acid (pABA) is not an essential nutrient for sulfonamide-resistant plasmodia. Instead, they seem to be able to synthesize pABA de novo. Four enzymes of the respective biosynthetic chain were demonstrated in isolated plasmodia: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase (EC 4.2.1.15), shikimate dehydrogenase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71) and pABA synthetase. We conclude that these three effects account for the reduced sulfonamide stress observed in the resistant parasite.

[Modern pancreatic function tests]. / Moderne Pankreasfunktionsdiagnostik.

The functional diagnostics is a corner-pillar of the difficult diagnostics of pancreas. Despite new tests many wishes remain open. The secretine-pancreozymine test and the Lundh-test give good informations, but they are expensive and for the patients considerably stressing. They certainly are not regarded as screening tests. The suitable and justifiable tests for epidemiologic examinations (estimations of stool enzymes and of serum isoamylase) are less specific and sensitive. A differentiation between chronic pancreatitis and neoplasm of the pancreas is not possible with the help of the functional diagnostics. The results of functional examinations may be correctly evaluated only within all informations which concentrate at the patient's bed.

[Clinical aspects and therapy of chronic pancreatitis]. / Klinik und Therapie der chronischen Pankreatitis.

Not infrequently exact diagnosis of chronic pancreatitis is possible only after step by step observation. The results of examination, analyses and treatment should be communicated from a general practitioner to a specialist, further to a specialized centre and vice versa. Screening tests and special examinations should be conducted in definite order. A surgical treatment is desirable in approximately 40% of clinically pronounced chronic pancreatitis. The best results are obtained by close cooperation between different specialists and the patient himself.

Regulation of a common amidotransferase subunit.

In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX). Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate. The trpX locus has been examined to determine its map position and control. (i) The trpX locus was found to be cotransformed with the lysS and pabA loci. The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA. (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein. (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci. (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus. These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus.