Purification and Characterization of Aminopeptidase B from Escherichia coli K-12

Aminopeptidase B, which is one of the four cysteinyl-glycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni²⁺, Mn²⁺, Co²⁺, and Cd²⁺, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidase B is a metallopeptidase. It was stabilized against heat in the presence of Mn²⁺ or Co²⁺. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The k_cat/K_m for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.     

Dihydroorotase from Escherichia coli. Sulfhydryl group-metal ion interactions

We have obtained 53 mg of 99% pure dihydroorotase from 10.9 g of frozen Escherichia coli pyrC plasmid-containing E. coli cells using a 4-step 16-fold purification procedure, a yield of 60%. We characterize the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (a dimer of subunit molecular weight 38,300 +/- 2,900), high performance liquid chromatography gel sieving, amino acid analysis, amino terminus determination (blocked), and specific activity. The isolated enzyme contains 1 tightly bound essential zinc atom/subunit, and readily but loosely binds 2 additional Zn(II) or Co(II) ions/subunit which modulate catalytic activity; treatment of crude extracts with weak chelators suggests that the enzyme contains 3 zinc atoms/subunit in vivo. Two of the 6 thiol groups/subunit react rapidly with 5,5'-dithiobis(2-nitrobenzoate) when 1 Zn/subunit enzyme is used, but slowly when 3 Zn/subunit enzyme is used. The 2 weakly bound Zn(II) ions/subunit protect against the reversible air oxidation which lowers the specific activity of the enzyme and renders it unreactive with 5,5'-dithiobis(2-nitrobenzoate). The dilution activation observed in the presence of substrate, the dilution inactivation observed in the absence of substrate, and the transient activation by the metal chelator oxalate are interpreted as evidence for an unstable, hyperactive monomer.     

MgATP and fructose 6-phosphate interactions with phosphofructokinase from Escherichia coli.

A thermodynamic linked-function analysis is presented of the interactions of MgATP and fructose 6-phosphate (Fru-6-P) with phosphofructokinase (PFK) from Escherichia coli in the absence of allosteric effectors. MgATP and Fru-6-P are shown to bind in random fashion by product inhibition of the back-reaction as well as by the kinetically competent binding of each ligand individually as monitored by the consequent changes in the intrinsic fluorescence of E. coli PFK. When Fru-6-P is saturating, the dissociation of MgATP is sufficiently slow that it cannot achieve a binding equilibrium in the steady state, causing the observed Km (49 microM) to significantly exceed the Kd (1.7 microM) deduced from a thermodynamic linkage analysis. The following features distinguish the interactions of MgATP and Fru-6-P with E. coli PFK: MgATP and Fru-6-P antagonize each other's binding to the enzyme in a saturable manner with an overall apparent coupling free energy equal to +2.5 kcal/mol at 25 degrees C; MgATP induces positive cooperativity in the Fru-6-P binding profile, with the Hill coefficient calculated from the Fru-6-P binding curves reaching a maximum of 3.6 when MgATP is saturating; and MgATP exhibits substrate inhibition at low concentrations of Fru-6-P. Simulations based upon the rate equation pertaining to a two-active-site, two-substrate dimer indicate that these features can all result from two independent couplings: an antagonistic MgATP-Fru-6-P coupling extending at least in part between active sites and a MgATP-induced Fru-6-P-Fru-6-P coupling.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new endoribonuclease from Escherichia coli. Ribonuclease N.

A new ribonuclease called RNase N was isolated from Escherichia coli. It is a nonspecific endoribonuclease that can cleave rRNA, poly(U), and poly(C) to small oligonucleotides and 5'-mononucleotides. It requires monovalent cations and is inhibited by divalent cations. It is suggested that this enzyme plays a role in the decay of rRNA,under various starvation conditions and perhaps in the decay of mRNA.     

RNA-protein interactions in the Escherichia coli ribosome.

Over the last two decades essentially three different approaches have been used to study the topography of RNA-protein interactions in the ribosome. These are: (a) the analysis of binding sites for individual ribosomal proteins or groups of proteins on the RNA; (b) the determination of protein footprint sites on the RNA by the application of higher order structure analytical techniques; and (c) the localisation of RNA-protein cross-link sites on the RNA. This article compares and contrasts the types of data that the three different approaches provide, and gives a brief and highly simplified summary of the results that have been obtained for both the 16S and 23S ribosomal RNA from E coli.     

Zinc interactions with regulatory dimers from Escherichia coli aspartate transcarbamoylase

Zn2+ is tetrahedrally bonded to the 4 nonadjacent thiols of each regulatory chain (Mr 17,000) near r-c contacts between catalytic (c) and regulatory chains (r) in aspartate transcarbamoylase (ATCase; c6r6). This paper reports on Zn2+ interactions with r dimer in the absence of stabilizing r-c contacts. After r2 and c3 subunits were separated, -SH groups of r2 were titrated with p-(hydroxymercuri)benzenesulfonate (PMPS) at pH 7.0. The concomitant release of Zn2+ (2 equiv/r dimer) was quantitated with 4-(2-pyridylazo)resorcinol (PAR) and was a linear function of PMPS added until 8 mercaptide bonds per r2 were formed. Breakage of 1 of 4 Zn2(+)-sulfur bonds in a Zn2+ binding cluster therefore makes the other three bonds more labile. From stopped-flow measurements, the PMPS-promoted Zn2+ release from r2 or mercaptide bond formation with 10- to 20-fold excess PMPS/r2-SH at pH 7.0 was first order with an Arrhenius activation energy Ea = 10 kcal/mol and a half-time t 1/2 = 9 +/- 2 ms at 20 degrees C without inhibitory anions present. The rate of mercurial-promoted Zn2+ release from r2 is at least 77 times faster than that from intact c6r6 [Hunt, J.B., Neece, S.H., Schachman, H.K., and Ginsburg, A. (1984) J. Biol. Chem. 259, 14793]; this indicates that Zn2+ binding clusters are more accessible to attack by PMPS than are those in ATCase. The addition of a 25-fold excess of the multidentate fluorescent chelator quin-2 to r2 gave a rate of Zn2+ dissociation that was 1/210th of that observed with excess mercurial. Furthermore, the Zn(PAR)1 complex was identified as the active species in the transfer of Zn2+ from Zn(PAR)2 to aporegulatory subunits, with kappa = (8 +/- 3) x 10(5) M-1 s-1 at pH 7.0 and 15 degrees C for this second-order association reaction. Although kinetic results are dependent on the mechanisms involved, an affinity constant K'A = (1.3 +/- 0.6) x 10(12) M-1 for Zn2+ binding to r dimer at pH 7.0 and 20 degrees C in the absence and presence of 100 mM KCl could be determined spectrally by rapid equilibration with the high-affinity, sensitive metalloindicators indo-1 and quin-2. This K'A value is based on the assumptions that Zn2+ binding sites in r2 are equivalent (noninteracting) and that apo-r2 does not dissociate; if apo-r2 dissociates, K'A approximately 10(14) M-1. Within experimental error, the K'A value was independent of [indo-1]/[r2] ratios from 36 to 3 with 0.3-8 microM r2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrostatic interactions of colicin E1 with the surface of Escherichia coli total lipid

The surface properties of colicin E1, a 522-amino acid protein, and its interaction with monolayers of Escherichia coli (E. coli) total lipid and 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DOPC) were studied using the Langmuir-Blodgett (LB) technique. Colicin E1 is amphiphilic, forming a protein monolayer at the air/buffer interface. The protein is thought to interact with the E. coli total lipid head groups through electrostatic interactions, followed by its insertion into the lipid monolayers. Supported lipid bilayers (SLBs) of E. coli total lipid and DOPC, deposited onto mica at the cell membrane equivalence pressure for E. coli and incubated with colicin E1, were imaged by contact mode atomic force microscopy (CM-AFM). Colicin E1 formed protein aggregates on DOPC SLBs, while E. coli total lipid SLB was deformed following its incubation with colicin E1. Corresponding lateral force images, along with electrostatic surface potentials for colicin E1 P190, imply a direct interaction of colicin E1 with lipid head groups facilitating their charge neutralization.     

Biochemical and topographical studies on Escherichia coli cell surface. IV. Giant spheroplast formation from a filamentous cell.

Long, nonseptate filamentous cells consisting of 5 to 40 single-cell unit lengths were formed from Escherichia coli surface mutant ONT-3 by treatment with a sublethal concentration of sodium dodecyl sylfate. As distinct from several other elongated cells (e.g., thymine-starved filaments), it was found here that stable giant spheroplasts, 5 to 10 micrometers in diameter, were produced by the action of lysozyme in the presence of bovine serum albumin via the gradual fusion of distinct spheroplasting bulbs.     

Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.     

Unusual synthesis by the Escherichia coli CCA-adding enzyme

The tRNA 3' end contains the conserved CCA sequence at the 74-76 positions. The CCA sequence is synthesized and maintained by the CCA-adding enzymes. The specificity of the Escherichia coli enzyme at each of the 74-76 positions was investigated using synthetic minihelix substrates that contain permuted 3' ends. Results here indicate that the enzyme has the ability to synthesize unusual 3' ends. When incubated with CTP alone, the enzyme catalyzed the addition of C74, C75, C76, and multiple Cs. Although the addition of C74 and C75 was as expected, that of C76 and multiple Cs was not. In particular, the addition of C76 generated CCC, which would have conflicted with the biological role of the enzyme. However, the presence of ATP prevented the synthesis of CCC and completely switched the specificity to CCA. The presence of ATP also had an inhibitory effect on the synthesis of multiple Cs. Thus, the E. coli CCA enzyme can be a poly(C) polymerase but its synthesis of poly(C) is regulated by the presence of ATP. These features led to a model of CCA synthesis that is independent of a nucleic acid template. The synthesis of poly(C) by the CCA-adding enzyme is reminiscent of that of poly(A) by poly(A) polymerase and it provides a functional rationale for the close sequence relationship between these two enzymes in the family of nucleotidyltransferases.     

In vivo cell division gene product interactions in Escherichia coli K-12.

Overexpression of plasmid-coded PBP 3 was analyzed in strains harboring ftsA, ftsH, pbpB (ftsI), ftsQ, ftsZ, or recA441 (Tif) mutations. Higher cellular levels of PBP 3, the pbpB gene product, could not restore septum formation of ftsA, ftsQ, ftsZ, and recA (Tif) mutants at 42 degrees C. However, filamentation in strains harboring pbpB and ftsH mutations was fully suppressed by PBP 3 overexpression. Additional observations indicated that the Y16 (ftsH) strain, not transformed with the PBP 3-overproducing plasmid, had no detectable PBP 3 in envelopes after incubation at the restrictive temperature. These results suggest that suppression of filamentation of fts strains overexpressing wild-type cell division proteins after the shift to the restrictive temperature can be a useful strategy to demonstrate in vivo interactions of cell division gene products.     

Unusual nucleotide arrangement with repeated sequences in the Escherichia coli K-12 chromosome.

Between 59 and 60 min on the Escherichia coli genetic map, there is a highly conserved sequence of 29 base pairs, containing an inverted repeat of seven base pairs that appears 14 times, 32 or 33 base pairs apart, downstream of the iap gene coding region. About 24 kilobase pairs downstream of the 14 repeats, a similar 29-base-pair sequence with a spacing of 32 base pairs appears seven times. Nucleotide sequences hybridizing with the 29-base-pair fragment were also detected in Shigella dysenteriae and Salmonella typhimurium but not in Klebsiella pneumoniae or Pseudomonas aeruginosa.     

Going for baroque at the Escherichia coli K1 cell surface

Phase variation is usually thought of as the stochastic switching between alternatively expressed ('on') and unexpressed ('off') phenotypic states. However, coupling synthesis of a monotonous homopolysaccharide to a mechanism of random but incomplete chemical modification produces almost infinite structural variation. Potentially limitless variability implies that evolution can produce highly ornate or extravagant flourishes reminiscent of the baroque style. Here, we describe an analysis of capsular polysialic acid form variation in Escherichia coli K1, demonstrating that the large number of variant structures is controlled by a single contingency locus. The mechanism for generating maximum structural diversity from maximal genetic parsimony is conferred by a simple translational switch carried on a K1-specific prophage.     

High-sensitivity detection of newly induced LamB protein on the Escherichia coli cell surface.

The kinetics of the appearance at the cell surface of the outer membrane LamB protein after induction were determined by using specific antibodies and radioiodinated protein A as a probe. This was done in two different induction systems. First, LamB protein was induced in a wild-type strain by the simultaneous addition of cyclic AMP and maltose. Second, an operon fusion strain in which the lamB gene is expressed under lac promoter control was used; in this system, LamB protein can be induced by isopropyl-beta-D-thiogalactopyranoside. When uninduced cells were grown in glucose minimal medium, background expression of the lamB gene was found to be ca. 10-fold lower in lac-lamB cells than in wild-type cells. The level of LamB protein present in uninduced wild-type cells could, however, be reduced by supplementing the growth medium with Casamino Acids. After induction, the LamB protein appeared at the cell surface of both strains within a few minutes, and then the LamB level per cell increased linearly. The time lag in cell surface exposure of LamB protein differed slightly under both induction conditions: the LamB protein appeared at the surface of lac-lamB cells within 3 min of induction, whereas in wild-type cells it could not be detected earlier than after 4 to 5 min of induction.     

Amino Acid Residues Involved in the Functional Integrity of Escherichia coli Methionine Aminopeptidase

Amino acid residues in the metal-binding and putative substrate-binding sites of Escherichia coli methionine aminopeptidase (MAP) were mutated, and their effects on the function of the enzyme were investigated. Substitution of any amino acid residue at the metal-binding site resulted in complete loss of the two cobalt ions bound to the protein and diminished the enzyme activity. However, only Cys70 and Trp221 at the putative substrate-binding site are involved in the catalytic activity of MAP. Changing either of them caused partial loss of enzyme activity, while mutations at both positions abolished MAP function. Both residues are found to be conserved in type I but not type II MAPs.     

Protein interactions in the outer membrane of Escherichia coli.

Specific protein interactions in Escherichia coli outer membrane were analyzed using chemical cross-linking with truly cleavable reagents and symmetrical two-dimensional sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The major outer membrane proteins were shown to form cross-linked complexes. These include multimers of lambda receptor, protein I, II, III and the free form of lipoprotein. Lipoprotein was also found to be cross-linked to proteins II and III. The identity of many of these complexes was verified using appropriate mutants missing the proteins in question. No new protein interactions were detected in the mutants even when three of the major proteins were missing. Proteins II, III and the free form of lipoprotein could also be cross-linked to the peptidoglycan layer of the cell wall.