Dissecting the molecular details of prokaryotic transcriptional control by surface plasmon resonance: the methionine and arginine repressor proteins.

The commercial surface plasmon resonance (SPR) biosensors, BIACORE, have been used to investigate the molecular details of macromolecular interactions at prokaryotic promoter-operators. For the Escherichia coli methionine repressor, MetJ, we have quantitated the interaction of the protein with synthetic and natural operator sites and shown that the SPR response is directly related to the stoichiometry of the complexes being formed. The utility of a continuous flow system has also been exploited to investigate transcription from an immobilised promoter-operator fragment; with transcripts collected and subsequently characterised by RT-PCR. This technique has enabled us to investigate how repressor binding affects (i) the interaction of the RNA polymerase (RNAP) with the promoter and (ii) the ability of RNAP to initiate transcription. Remarkably, the repression complex appears to stabilise binding of RNAP, whilst having the expected effects on the levels of transcripts produced. This may well be a general mechanism allowing rapid transcription initiation to occur as soon as the repression complex dissociates. These techniques have also been used to examine protein-DNA interactions in the E. coli and Bacillus subtilis arginine repressor systems. The repressors are the products of the argR and ahrC genes, respectively. Both proteins form hexamers in rapid equilibrium with smaller subunits believed to be trimers. There are three types of operator in these systems, autoregulatory, biosynthetic and catabolic (B. subtilis only). Sensorgrams show that each protein recognises the three types of immobilised operator differently and that binding is stimulated over 100-fold by the presence of L-arginine.

Structure and mechanism of galactose oxidase. The free radical site.

Crystallographic and spectroscopic studies on galactose oxidase have shown that the active site involves a free radical on tyrosine 272, one of the ligands coordinated to the Cu2+ cofactor. A novel thioether bond between tyrosine 272 and cysteine 228, and a stacking tryptophan 290, over this bond, are features of the crystal structure. The present study describes the development of a high level heterologous expression system for galactose oxidase and the construction of mutational variants at these key active site residues. The expressed wild-type enzyme and mutational variants (W290H and C228G) have been characterized by x-ray crystallography, visible spectroscopy, and catalytic activity measurements. A further variant protein, Y272F, could not be purified. The data establish that the thioether bond and stacking tryptophan are essential for activity and further support a role for tryptophan 290 as a component of the free radical site.

Alginate lyase from Klebsiella pneumoniae, subsp. aerogenes: gene cloning, sequence analysis and high-level production in Escherichia coli.

The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25, has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa, in good agreement with SDS-PAGE analysis, and a calculated pI of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus, probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse a beta-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.

The gdhA1 point mutation in Escherichia coli K12 CLR207 alters a key lysine residue of glutamate dehydrogenase.

gdhA1 is a spontaneous mutant of Escherichia coli that causes complete loss of activity of the NADP-specific glutamate dehydrogenase (GDH) encoded by the gdhA gene. The gdhA1 mutational site has been identified by recombinational mapping, polymerase chain reaction (PCR) amplification and DNA sequencing, as an A to G transition at nucleotide 274 of the gdhA coding sequence, resulting in an amino acid change of lysine 92 to glutamic acid. The mutant enzyme forms hybrid hexamers with a wild-type GDH, providing a useful system for analysis of conformational integrity of mutational variants.

A nitrate reductase gene of the cyanobacterium Synechococcus PCC6301 inferred by heterologous hybridization, cloning and targeted mutagenesis.

DNA probes from the narG gene of Escherichia coli, which encodes the large polypeptide of respiratory nitrate reductase, show cross-hybridization at low stringency to a single region of the genome of the cyanobacterium Synechococcus PCC6301. This segment of cyanobacterial DNA was cloned as the insert of plasmid pDN1 and characterized. RNA complementary to pDN1 was shown to be substantially more abundant in nitrate grown cells of Synechococcus PCC6301 than in ammonium grown cells, thus parallelling the nitrate induction and ammonium repression of nitrate reductase activity in cultures of this cyanobacterium. A mutant of Synechococcus PCC6301 deficient in nitrate reductase activity was obtained after a potentially mutagenic transformation treatment using pDN1 as a donor. This mutant was restored to the wild type phenotype following stable integrative transformation with pDN1 DNA. Taken together these data suggest that pDN1 might encode a polypeptide of nitrate reductase. pDN1 is distinct from three clones of genes involved in nitrate assimilation that were isolated previously from the related cyanobacterium Synechococcus PCC7942 (Kuhlemeier et al., 1984a, J. Bact. 159, 36-41, and 1984b, Gene 31, 109-116).

Multiple interactions of lysine-128 of Escherichia coli glutamate dehydrogenase revealed by site-directed mutagenesis studies.

A highly conserved lysine at position 128 of Escherichia coli glutamate dehydrogenase (GDH) has been altered by site-directed mutagenesis of the gdhA gene. Chemical modification studies have previously shown the importance of this residue for catalytic activity. We report the properties of mutants in which lysine-128 has been changed to histidine (K128H) or arginine (K128R). Both mutants have substantially reduced catalytic centre activities and raised pH optima for activity. K128H also has increased relative activity with amino acid substrates other than glutamate, especially L-norvaline. These differences, together with alterations in Km values, Kd values for NADPH and Ki values for D-glutamate, imply that lysine-128 is intimately involved in either direct or indirect interactions with all the substrates and also in catalysis. These multiple interactions of lysine-128 explain the diverse effects of chemical modifications of the corresponding lysine in homologous GHDs. In contrast, lysine-27, another highly reactive residue in bovine GDH, is not conserved in all of the sequenced NADP-specific GDHs and is therefore not likely to be involved in catalysis.

Expression of the Escherichia coli glutamate dehydrogenase gene in the cyanobacterium Synechococcus PCC6301 causes ammonium tolerance.

The unicellular cyanobacterium Synechococcus PCC6301 lacks a hybridisable homologue of the strongly conserved gdhA gene of E. coli that encodes NADP-specific glutamate dehydrogenase. This is consistent with the failure to find this enzyme in extracts of the cyanobacterium. The E. coli gdhA gene was transferred to Synechococcus PCC6301 by transformation with an integrative vector. High levels of glutamate dehydrogenase activity, similar to those found in ammonium grown E. coli cells, were found in these transformants. These transformed cyanobacteria displayed an ammonium tolerant phenotype, consistent with the action of their acquired glutamate dehydrogenase activity as an ammonium detoxification mechanism. Minor differences in colony size and in growth at low light intensity were also observed.

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli.

The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.

Respiratory nitrate reductase of Escherichia coli. Sequence identification of the large subunit gene.

The nucleotide sequence is presented of part of the transcription initiation end of the nar operon of Escherichia coli K12, which encodes the respiratory nitrate reductase complex. The first coding sequence transcribed is the narG gene, encoding the large catalytic molybdoprotein of the complex. This sequence was assigned unambiguously by automated N-terminal amino acid sequencing of the purified large subunit. The deduced partial amino acid sequence of this polypeptide is hydrophilic and rich in basic residues. Membrane insertion does not involve N-terminal proteolytic processing of this subunit.

Amino acid replacements resulting from suppression and missense reversion of a chain-terminator mutation in Neurospora.

The Neurospora crassa super-suppressor mutation, ssu-1, suppresses the auxotrophic phenotype of the mutant am(17) by inserting tyrosine at residue 313 of NADP-specific glutamate dehydrogenase, a position occupied in the wild type by glutamate. Two classes of am(17) revertants due to further mutation within the am gene have, respectively, tyrosine and leucine at residue 313. These replacements are consistent with a chain-terminating codon in am(17) of either the amber (UAG) or the ochre type (UAA), but are inconsistent with UGA. The Leu313 and Tyr313 variants of the enzyme have effective activity but are grossly different from the wild type in Michaelis constants (especially for ammonium) and heat stabilities at two different pH values. They show smaller but significant differences in these respects from each other.

Re-investigation of the effects of L-glutamine and L-asparagine on the Neurospora crassa NADP-specific glutamate dehydrogenase.

L-Glutamine, when purified free of traces of NH4+ present in solution, does not act as an alternative substrate to NH4+ for the NADP-specific glutamate dehydrogenase of Neurospora. L-Glutamine interferes with detection of small quantities of NH4+ by Nessler's reagent. L-Asparagine is not an alternative substrate to NH4+ for this enzyme.

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptides from digestion with a staphylococcal proteinase.

The extracellular proteinase of Staphylococcus aureus strain V8 was used to digest the NADP-specific glutamate dehydrogenase of Neurospora crassa. Of 35 non-overlapping peptides expected from the glutamate content of the polypeptide chain, 29 were isolated and substantially sequenced. The sequences obtained were valuable in providing overlaps for the alignment of about two-thirds of the sequences found in tryptic peptides [Wootton, J. C., Taylor, J, G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 739-748]. The blocked N-terminal peptide of the protein was isolated. This peptide was sequenced by mass spectrometry, and found to have N-terminal N-acetylserine by Howard R. Morris and Anne Dell, whose results are presented as an Appendix to the main paper. The staphylococcal proteinase showed very high specificity for glutamyl bonds in the NH4HCO3 buffer used. Partial splits of two aspartyl bonds, both Asp-Ile, were probably attributable to the proteinase. No cleavage of glutaminyl or S-carboxymethylcysteinyl bonds was found. Additional experimental detail has been deposited as Supplementary Publication SUP 50053 (5 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K, from whom copies may be obtained under the terms given in Biochem. J. (1975) 1458 5.

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptic and chymotryptic peptides and the complete sequence.

Peptic and chymotryptic peptides were isolated form the NADP-specific glutamate dehydrogenase of Neurospora crassa and substantially sequenced. Out of 452 residues in the polypeptide chain, 265 were recovered in the peptic and 427 in the chymotryptic peptides. Together with the tryptic peptides [Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 749-755], these establish the complete sequence of the chain, including the acid and amide assignments, except for seven places where overlaps are inadequate. These remaining alignments are deduced from information on the CNBr fragments obtained in another laboratory [Blumenthal, K. M., Moon, K. & Smith, E. L. (1975), J. Biol. Chem. 250, 3644-3654]. Further information has been deposited as Supplementary Publication SUP 50054 (17 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1975) 145, 5.

Amino-acid sequence of NADP-specific glutamate dehydrogenase of neurospora crassa.

A tentative primary structure of the NADP-specific glutamate dehydrogenase [L-glutamate: NADP oxidoreductase (deaminating), EC 1.4.1.4] from Neurospora crassa has been determined. The proposed sequence contains 452 amino-acid residues in each of the identical subunits of the hexameric enzyme. Comparison of the sequence with that of the bovine liver enzyme reveals considerable homology in the amino-terminal portion of the chain, including the vicinity of the reactive lysine, with only shorter stretches of homology within the carboxyl-terminal regions. The significance of this distribution of homologous regions is discussed.