Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.     

Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.     

Zinc binding by the methylation signaling domain of the Escherichia coli Ada protein.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.     

In vitro proteolytic cleavage of the Escherichia coli Ada protein by the ompT gene product.

Down regulation of the adaptive response to alkylation damage in Escherichia coli has been proposed to occur by proteolytic cleavage of the regulatory Ada protein. In this paper, it is shown that proteolysis of the Ada protein as observed in cell extracts is caused by the ompT gene product. This protease, however, was not involved in switching off the adaptive response in vivo.     

The structure and mechanism of serine acetyltransferase from Escherichia coli

Serine acetyltransferase (SAT) catalyzes the first step of cysteine synthesis in microorganisms and higher plants. Here we present the 2.2 A crystal structure of SAT from Escherichia coli, which is a dimer of trimers, in complex with cysteine. The SAT monomer consists of an amino-terminal alpha-helical domain and a carboxyl-terminal left-handed beta-helix. We identify His(158) and Asp(143) as essential residues that form a catalytic triad with the substrate for acetyl transfer. This structure shows the mechanism by which cysteine inhibits SAT activity and thus controls its own synthesis. Cysteine is found to bind at the serine substrate site and not the acetyl-CoA site that had been reported previously. On the basis of the geometry around the cysteine binding site, we are able to suggest a mechanism for the O-acetylation of serine by SAT. We also compare the structure of SAT with other left-handed beta-helical structures.     

Protease So from Escherichia coli preferentially degrades oxidatively damaged glutamine synthetase

After oxidative damage (e.g. induced with iron, ascorbate, and oxygen), the inactivated glutamine synthetase is selectively hydrolyzed in extracts of Escherichia coli. We therefore tested if glutamine synthetase treated with this system is hydrolyzed preferentially by any of the known E. coli proteases. Protease So, a cytoplasmic serine protease, was found to degrade the oxidized form of glutamine synthetase to acid-soluble peptides 5-10 times faster than the native glutamine synthetase. Degradation of the oxidized glutamine synthetase was inhibited by EDTA and stimulated 5-10-fold by Mg2+, Ca2+, or Mn2+, even though casein hydrolysis by protease So is not affected by divalent cations. Apparently, these cations affect the conformation of this substrate, making it more susceptible to proteolytic attack. Protease Re, another cytoplasmic protease, also degrades preferentially the oxidized form of glutamine synthetase and seems to correspond to the glutamine synthetase-degrading activity recently described by Roseman and Levine [1987) J. Biol. Chem. 262, 2101-2110). However, it is much less active in this reaction than protease So. No other soluble E. coli protease, including Do, Ci, Mi, Fa, Pi, or the ATP-dependent proteases Ti and La (the lon product), appears to degrade this oxidized protein. These results suggest that protease So participates in the hydrolysis of oxidatively damaged proteins and that E. coli has multiple systems for degrading different types of aberrant proteins.     

Target of serine inhibition in Escherichia coli

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.     

Random-order ternary complex reaction mechanism of serine acetyltransferase from Escherichia coli

Although serine acetyltransferase (SAT) from Escherichia coli is homologous with a number of bacterial enzymes that catalyze O-acetyl transfer by a sequential (ternary complex) mechanism, it has been suggested, from experiments with the nearly identical enzyme from Salmonella typhimurium, that the reaction could proceed via an acetyl-enzyme intermediate. To resolve the matter, the E. coli gene for SAT was overexpressed and the enzyme purified 13-fold to homogeneity. The results of a steady-state kinetic analysis of the forward reaction are diagnostic for a ternary complex mechanism, and the response of SAT to dead-end inhibitors indicates a random order for the addition of substrates. The linearity of primary double-reciprocal plots, in the presence and absence of dead-end inhibitors, argues that interconversion of ternary complexes is not significantly faster than kcat, whereas substrate inhibition by serine suggests that breakdown of the SAT.CoA binary complex is rate-determining. The results of equilibrium isotope exchange experiments, for both half-reactions, rule out a "ping-pong" mechanism involving an acetyl-enzyme intermediate, and a pre-steady-state kinetic analysis of the turnover of AcCoA supports such a conclusion. Kinetic data for the reverse reaction (acetylation of CoA by O-acetylserine) are also consistent with a steady-state random-order mechanism, wherein both the breakdown of the SAT*serine complex and the interconversion of ternary complexes are partially rate-determining.     

Inhibition of RecA protein function by the RdgC protein from Escherichia coli

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.     

Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42 degrees C in the presence of a lac inducer, isopropyl-beta-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.     

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.     

Purification of thiamine-binding protein from Escherichia coli

An affinity column coupled with thiamine pyrophosphate quantitatively absorbs the thiamine-binding activity from a partially purified preparation of $\text{Escherichia}\text{coli}$. The thiamine-binding protein can be eluted from the affinity column in high yield by use of 8 M urea-containing buffer. Approximately 90-fold purification occurs by affinity chromatography yielding a preparation which appears to be homogenous.