Properties of xanthosine 5'-monophosphate-amidotransferase from Escherichia coli.

Xanthosine 5′-phosphate (XMP)-amidotransferase catalyzes the formation of guanosine 5′-phosphate (GMP) by aminating XMP with either the amide group of glutamine (amidotransferase) or ammonia (aminase). The glutamine-supported activity of the purified enzyme from Escherichia coli has been studied, and its properties have been compared with those of other amidotransferases. The following results have been obtained. (i) The glutamine analog, 6-diazo-5-oxo-l-norleucine (DON), irreversibly inhibits the amidotransferase activity. A maximal rate of inhibition by DON is achieved in the presence of XMP, ATP, and Mg²⁺ with a pseudo-first-order rate constant of 0.276 min^?1. (ii) The total number of sulfhydryl groups is approximately 22 per dimer (126,000 M_r). In the absence of substrates, about 8 sulfhydryl groups per dimer are titratable with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), and in the presence of XMP, ATP, and Mg²⁺ an additional 6 cysteine residues per dimer become exposed. When the amidotransferase activity is inactivated by DON, only 8 sulfhydryl groups are titratable. DTNB, p-chloromercuribenzoate, and bromopyruvate all selectively inactivate the amidotransferase activity. These results are consistent with the hypothesis that cysteine residues which are exposed by the substrates are involved in the amidotransferase activity. (iii) The purified XMP amidotransferase contains a glutaminase activity which can be measured in the absence of GMP formation. The glutaminase activity requires XMP, Mg²⁺, and either psicofuranine, an analog of adenosine, or inorganic pyrophosphate (PP_i) and is inhibited by p-chloromercuribenzoate and DON. Maximal stimulation is observed with 100 μm psicofuranine or PP_i, and there is no further stimulation in the presence of both effectors. The apparent K_m is 31 μm with PP_i and 13 μm with psicofuranine; the V for glutamine hydrolysis is about 60% of the rate of the amidotransferase activity. The cooperative interactions between the binding of PP_i and psicofuranine have been confirmed. In the presence of 2.5 μm psicofuranine the K_m for PP_i is reduced 20-fold, but the maximal velocity is unchanged. Similarly, the apparent K_m for psicofuranine is reduced by low concentrations (10 μm) of PP_i. The “uncoupling” of the hydrolysis of glutamine from the amination of XMP is the basis for the reported inhibitory effects of psicofuranine and PP_i on the amidotransferase activity. (iv) Tris buffer selectively inhibits the XMP-amidotransferase activity by inhibiting the glutaminase activity. This inhibition is time dependent and reversible and may explain the previous reports on the inability of this enzyme to use glutamine as a substrate.     

The kinetics of Schiff-base formation during reconstitution of D-serine apodehydratase from Escherichia coli with pyridoxal 5'-phosphate.

Schiff base formation during reconstitution of D-serine dehydratase (Escherichia coli) from its apoenzyme and pyridoxal 5'-phosphate (pyridoxal-P) has been studied by rapid kinetic techniques using absorbance changes at 436 nm. Three distinct reaction phases have been observed. The first is a very rapid change during which pyridoxal-P is initially bound to the apoenzyme. This step has an equilibrium constant of 1500 M-1 and a forward reaction rate of the order of 2.6 x 10(6) M-1 s-1. The second phase shows a first-order rate constant with a value dependent on pyridoxal-P and corresponds to a first-order step with a forward rate constant of 3.04 s-1 interacting with the initial equilibrium. The final phase is a slow first-order reaction, the rate constant of which is approximately 0.01 s-1 and is independent of pyridoxal-P concentration. The active pyridoxal species has been shown to be the free pyridoxal-P as opposed to hemiacetal or hemimercaptal forms.     

dnaC-dependent reconstitution of replication forks in Escherichia coli lysates.

Lysates of Escherichia coli exhibit a DNA-synthesizing activity that depends on the presence of replication forks and of replication proteins. Replicative activity was reconstituted in vitro by mixing lysates prepared from temperature-sensitive dnaB mutants with wild-type dnaB protein. Lysates of double mutants deficient in both dnaB and dnaC genes could only be complemented by the addition of both dnaB and dnaC proteins, whereas lysates deficient in dnaC protein did not require the addition of any exogenous factor. This shows that the replication machinery, once it is running along the chromosome, is independent of dnaC protein, dnaC activity, however, is required for the replacement of defective dnaB protein at running replication forks.     

Conditional synthesis and utilization of 1,5-anhydroglucitol in Escherichia coli.

A cyclic polyol, 1,5-anhydro-D-glucitol (AG), is widely detected in most organisms, although little is known about its metabolism and physiological roles. The present study demonstrates the synthesis of AG in Escherichia coli C600. The major portion of the synthesized AG was indicated to be derived from glucose retaining all the six carbon atoms, and only 5% was attributed to AG synthesized from C3 compounds. AG synthesis is apparent in an early stage of the stationary phase, and accumulation is transient both in cells and in medium. Evidence is also presented for AG uptake and metabolism and for effects of cyclic AMP.     

Properties of Escherichia coli mutants with alterations in Mg2+-adenosine triphosphatase.

A mutant Escherichia coli, selected for resistance to the antibiotic neomycin, was unable to utilize nonfermentable carbon sources for growth. Two strains were selected from this mutant on the basis of their ability to grow utilizing succinate as a carbon source. All three strains had approximately equal amounts of the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) protein, but the activity of the enzyme differed in each strain. The Mg2+-ATPase from each of the three strains lost activity upon solubilization and appeared to undergo rapid dissociation once solubilized. This dissociation is similar to that described for the wild type after cold exposure.     

In vitro reconstitution of catabolite repression in Escherichia coli

A widely accepted model for catabolite repression posits that phospho-IIAGlc of the bacterial phosphotransferase system activates adenylyl cyclase (AC) activity. For many years, attempts to observe such regulatory properties of AC in vitro have been unsuccessful. To further study the regulation, AC was produced fused to the transmembrane segments of the serine chemoreceptor Tsr. Cells harboring Tsr-AC and normal AC, expressed from the cya promoter on a low copy number vector, exhibit similar behavior with respect to elevation of cAMP levels resulting from deletion of crp, expressing the catabolite regulatory protein. Membrane-bound Tsr-AC exhibits activity comparable with the native form of AC. Tsr-AC binds IIAGlc specifically, regardless of its phosphorylation state, but not the two general phosphotransferase system proteins, enzyme I and HPr; IIAGlc binding is localized to the C-terminal region of AC. Binding to membranes of either dephospho- or phospho-IIAGlc has no effect on AC activity. However, in the presence of an Escherichia coli extract, P-IIAGlc, but not IIAGlc, stimulates AC activity. Based on these findings of a direct interaction of IIAGlc with AC, but activity regulation only in the presence of E. coli extract, a revised model for AC activity regulation is proposed.     

Simultaneous reconstitution of Escherichia coli membrane vesicles with D-lactate and D-amino acid dehydrogenases

Purified preparations of D-amino acid dehydrogenase [Olsiewski, P.J., Kaczorowski, G. J., & Walsh, C. T. (1980) J. Biol. Chem. 225, 4487] and D-lactate dehydrogenase [Kohn, L.D., & Kaback, H.R. (1973) J. Biol. Chem. 248, 7012] bind independently to right-side-out and inverted Escherichia coli vesicles and to phosphatidylcholine liposomes without detectable competition. The reconstituted vesicles catalyze D-lactate- and D-alanine-dependent respiration (O2 uptake), proton translocation, and proton/lactose symport. The enzymes do not share common sites of association on either face of the E. coli membrane, and binding of both enzymes to the bilayer appears to be due to nonspecific affinity for the surface rather than specific binding to proteinaceous receptors. Each enzyme, however, appears to reduce a common proton translocating step in the membrane-bound respiratory chain, and substrate-derived electrons are transferred through a common rate-determining redox component that precedes the site of proton translocation. The results suggest that although binding is nonspecific, there is a common site for proton translocation in the membrane between the flavin-linked dehydrogenases and the cytochromes and that this site is accessible by distinct routes of electron transfer from primary dehydrogenases on either surface of the membrane.     

Properties of deoxynucleoside 5''-monophosphatase induced by bacteriophage T5 after infection of Escherichia coli.

Bacteriophage T5 induced a deoxynucleoside 5'-monophosphatase during its infection of Escherichia coli. The enzyme was purified about 100-fold. It was clearly distinct from the host 5'-nucleotidase activity in its physical characteristics and substrate specificity. The enzyme was active on deoxynucleoside 5'-monophosphates but was not active as a phosphatase on ribonucleotides, deoxynucleoside 5'-triphosphates, deoxynucleoside 3'-monophosphates, or deoxyoligonucleotides. Furthermore, it did not have oligonucleotidase or exonuclease activity. The enzyme could exist in multimeric form but had a monomer molecular weight of about 25,000.     

In vitro reconstitution of anticodon nuclease from components encoded by phage T4 and Escherichia coli CTr5X.

During phage T4 infection of Escherichia coli strains containing the prr locus the host tRNALys undergoes cleavage-ligation in reactions catalyzed by anticodon nuclease, polynucleotide kinase and RNA ligase. Known genetic determinants of anticodon nuclease are prr, which restricts T4 mutants lacking polynucleotide kinase or RNA ligase, and stp, the T4 suppressor of prr encoded restriction. The present communication describes an in vitro anticodon nuclease assay in which the specific cleavage of tRNALys is driven by an extract from E. coli prrr (restrictive) cells infected by phage T4. The in vitro anticodon nuclease reaction requires factor(s) encoded by prr, is stimulated by a synthetic Stp polypeptide and appears to require additional T4 induced factor(s) distinct from Stp.     

Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels.

We have modified the procedure of Criado and Keller (1987) to study ion channels of Escherichia coli reconstituted in liposomes. The modifications include (a) excluding the use of any detergent and (b) inducing blisters from liposomes with Mg2+. These blisters, which appear to be unilamellar, are stable for hours. They could be repeatedly sampled with different patch-clamp pipettes each achieving seal resistance greater than 10 GOhms. Activities of three types of ion channels are often observed by use of this method, including two voltage-sensitive cation channels of different conductances. Even the mechanosensitive channel, previously recorded from live E. coli cells (Martinac et al., 1987), was also detected in these blisters. Apparently the channel protein and any accessory structures, postulated to be needed for mechanotransduction, can be reconstituted together by this method.