The structure of E. coli beta-galactosidase

E. coli beta-galactosidase is a tetramer of four identical 1023-amino acid chains. Each chain consists of five domains, the third of which is an eight-stranded alpha/beta barrel that comprises much of the active site. This site does, however, include elements from other domains and other subunits. The N-terminal region of the polypeptide chains help form one of the subunit interfaces. Taken together these features provide a structural basis for the well-known property of alpha-complementation. Catalytic activity proceeds via the formation of a covalent galactosyl intermediate with Glu537, and includes 'shallow' and 'deep' modes of substrate binding.     

The action of beta-galactosidase (Escherichia coli) on allolactose.

The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.     

Dihydrofolate reductase (mouse) and beta-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E. coli

Hybrid genes were constructed. One, ompA153-dfr, encoded the precursor of the 325 residue Escherichia coli outer membrane protein OmpA up to residue 153 which was fused to the complete 186-residue dihydrofolate reductase of the mouse. The other, ompA219-lacZ, coded for the same precursor up to residue 219 which was fused to 1017 COOH-terminal residues of the 1023-residue subunit of the beta-galactosidase of E. coli. Full expression of the ompA153-dfr gene caused accumulation of its precursor and of that of the chromosomally encoded OmpA protein. When the amount of product was reduced, no pro-OmpA and very little pro-hybrid protein accumulated. The precursor was processed and the mature protein was fully accessible to trypsin in permeabilized cells. Expression of the ompA219-lacZ gene led to the presence of the hybrid protein at only 20-30% of the amount expected. About 20% of it appeared to be incorporated in the outer membrane. All of the hybrid was quantitatively accessible to trypsin in permeabilized cells. When the hybrid gene was overexpressed, the protein was found associated with the plasma membrane in the cytosol. It is concluded that both beta-galactosidase and dihydrofolate reductase could quantitatively traverse the plasma membrane, provided the amounts synthesized were sufficiently small.     

Immobilization of E. coli beta-galactosidase and its derivatives by polyacrylamide gel

We have recently prepared some crosslinked derivatives of Escherichia coli beta-galactosidase by treating the enzyme with bisimidoesters. In this article, we report the results obtained when the native and these crosslinked derivatives are entrapped in polyacrylamide gel lattice. It was found that use of combination of three protective agents, viz., bovine serum albumin, cysteine, and lactose, during immobilization gave an increased yield of 190% in the case of DMA crosslinked preparation. In the case of native enzyme, the K(m), pH optimum, and temperature optimum were found to remain unchanged on immobilization. The DMA crosslinked preparation entrapped in polyacrylamide in the presence of BSA, lactose, and cysteine was found to be a significantly better catalyst and hydrolyzed 47% milk lactose as compared to 31% hydrolysis by entrapped native enzyme in 6 h.     

Stimulation of the differential rate of beta-galactosidase synthesis in E. coli by trimethoprim

Summary It was found that partial inhibition of peptide chain initiation by trimethoprim causes a significant increase in the differential rate of -galactosidase in cultures of E. coli growing in the presence of suboptimal concentrations of a galactoside inducer. This stimulation is not produced by inhibitors of peptide chain growth, such as chloramphenicol and puromycin. Trimethoprim, furthermore, does not cause a significant increase in the differential rate of -galactosidase synthesis in E. coli cultures (a) of i ⁺ genotype which are fully induced, (b) of fully constitutive i ^- genotype, (c) of partially constitutive o ^c genotype, or (d) of noninducible i ^s genotype. These findings are compatible with the idea that the differential rate of -galactosidase synthesis is regulated by means of an inducer-dependent growth period of a regulatory polypeptide.     

Targeting of E. coli beta-galactosidase to the nucleus in yeast

In order to identify determinants governing nuclear protein localization, we constructed a set of hybrid genes by fusing the S. cerevisiae gene, MAT alpha 2, coding for a presumptive nuclear protein, and the E. coli gene, lacZ, coding for beta-galactosidase. The resultant hybrid proteins contain 3, 13, 25, 67, or all 210 amino acids of wild-type alpha 2 protein at the amino terminus and a constant, enzymatically active portion of beta-galactosidase at the carboxy terminus. Indirect immunofluorescence and subcellular fractionation studies with yeast cells containing the alpha 2-LacZ hybrid proteins indicate that the alpha 2 segment can direct localization of beta-galactosidase to the nucleus. A segment as small as 13 amino acids from alpha 2 is sufficient for this localization. Comparison of amino acid sequences of other nuclear proteins with this region of alpha 2 reveals a sequence that may be necessary for nuclear targeting. Production of some alpha 2-LacZ hybrid proteins causes cell death, perhaps as a result of improper or incomplete localization. These studies also indicate that the alpha 2 protein, argued on genetic grounds to be a negative regulator, acts in the yeast nucleus.     

Site-directed mutagenesis of beta-galactosidase (E. coli) reveals that tyr-503 is essential for activity

By using the technique of site-directed mutagenesis we have succeeded in replacing tyr-503 of beta-galactosidase (E. coli) with a phe. A study of the kinetic and stability properties of this mutant enzyme (F-503 beta-galactosidase) showed that the loss in activity upon this change is due to the loss of a catalytic group (rather than a detrimental change in the enzyme's overall structure or a change in the enzyme's binding capacity). This confirms previous suggestions that this tyr residue is involved in catalysis.     

Plasmid vectors with a semi-synthetic beta-galactosidase gene of E. coli

A partial synthesis of a structural gene for beta-galactosidase and construction of a series of pLZ plasmids for quantitative study of E. coli promoters are reported. The gene was assembled of two short synthetic DNAs and of a 3000 bp long EcoRI fragment (comprising the lacZ sequence 16-3013) isolated from plasmid p198/1 of B. Gronenborn. Among the plasmids constructed, pLZ4 is a promoter-probe vector that contains the semi-synthetic gene fused with a synthetic Shine-Dalgarno sequence and preceded by unique EcoRI and KpnI cleavage sites. On cloning a promoter into these sites, its signal strength in vivo could easily be measured by assaying beta-galactosidase activity. The use of pLZ4 vector was demonstrated by quantifying the effect of T7 early promoters A1 and A2, the latter being found 4,5 times more active under the conditions employed.     

A quantitation of the factors which affect the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) on lactose.

A study was implemented to quantitate the hydrolase and transgalactosylase activities of beta-galactosidase (E. coli) with lactose as the substrate and to investigate various factors which affect these activities. At low lactose concentrations the rate of galactose production was equal to the rate of glucose production. The rate of galactose production relative to glucose, however, dropped dramatically at lactose concentrations higher than 0.05 M and production of trisaccharides and tetrasaccharides began (galactose/glucose ratios of about 2:1 and 3:1, respectively, were found for these two types of oligosaccharides). At least five different trissacharides were formed and their patterns of formation showed that they probably utilized both lactose and allolactose as galactosyl acceptors. Allolactose was produced in amounts proportional to glucose at all lactose concentrations (ratios of allolactose/glucose were about 0.88). Analyses of various data, including a reaction analyzed at very early times, showed that the major means of production of allolactose (and the only means initially) was the direct enzymatic transfer of galactose from the 4 position to the 6 position of the glucose moiety of lactose without prior release of glucose from the enzyme. It was shown, however, that allolactose could also be formed in significant quantities by the transfer of galactose to the 6 position of free glucose, and also by hydrolysis of preformed trisaccharide. A mechanism which fits the initial velocity data was proposed in which the steps involving the formation of an enzyme-gallactose-glucose complex, the formation and breakage of allolactose on the enzyme, and the release of glucose all seem to be of roughly equal magnitude and rate determining. Various factors affected the amounts of transgalactosylase and hydrolase activities occurring. At high pH values (greater than 7.8) the transgalactosylase/hydrolyase activity ratio increased dramatically while it decreased at low pH values (less than 6.0). At mid pH values the ratio was essentially constant. The absence of Mg2+ caused a large decrease in the transgalactosylase/hydrolase activity ratio while the absence of all but traces of Na+ or K+ had no effect. The anomeric configuration of lactose altered the transgalactosylase/hydrolase activity ratios, alpha-Lactose resulted in a decrease of allolactose production (transgalactosylase activity) relative to hydrolase activities (glucose production) while beta-lactose had the opposite effect.     

Engineering the E. coli beta-galactosidase for the screening of antiviral protease inhibitors

Site-specific proteolysis is essential in many fundamental cellular and viral processes. It has been previously shown that the Escherichia coli beta-galactosidase can be useful for the high-throughput screening of human immunodeficiency virus type 1 protease inhibitors. Here, by using crystallographic and functional data of the bacterial enzyme, we have identified a new accommodation site between amino acids 581 and 582, in a solvent-exposed and flexible beta-turn of domain III. The placement of the model peptide reproducing the matrix-capsid (p17/p24) gag cleavage sequence renders a highly active and efficiently digested chimeric construct. The use of this insertion site, that increases the cleavage potential of this reporter enzyme, can improve the sensitivity and dynamic range of the antiviral drug assay. This simple and highly specific analytical test may also be extended to the screening of other specific protease inhibitors by a convenient colorimetric assay.     

A structural view of the action of Escherichia coli (lacZ) beta-galactosidase.

The structures of a series of complexes designed to mimic intermediates along the reaction coordinate for beta-galactosidase are presented. These complexes clarify and enhance previous proposals regarding the catalytic mechanism. The nucleophile, Glu537, is seen to covalently bind to the galactosyl moiety. Of the two potential acids, Mg(2+) and Glu461, the latter is in better position to directly assist in leaving group departure, suggesting that the metal ion acts in a secondary role. A sodium ion plays a part in substrate binding by directly ligating the galactosyl 6-hydroxyl. The proposed reaction coordinate involves the movement of the galactosyl moiety deep into the active site pocket. For those ligands that do bind deeply there is an associated conformational change in which residues within loop 794-804 move up to 10 A closer to the site of binding. In some cases this can be inhibited by the binding of additional ligands. The resulting restricted access to the intermediate helps to explain why allolactose, the natural inducer for the lac operon, is the preferred product of transglycosylation.     

Suicide inactivation of the E. coli O6-methylguanine-DNA methyltransferase

The O6-methylguanine-DNA methyltransferase of Escherichia coli acts rapidly and stoichiometrically to convert a mutagenic O6-methylguanine residue in DNA to unsubstituted guanine. Even at low protein concentrations and in the absence of any cofactors, the transfer of a methyl group to one of the protein's own cysteine residues occurs in less than 2 s at 37 degrees C. The entire kinetic process can be followed experimentally at 5 degrees C. Formation of S-methylcysteine in the protein is accompanied by loss of activity and accounts for the exceptional suicide kinetics of this enzyme as well as for the sharp saturation of O6-methylguanine repair observed in vivo. The enzyme can remove greater than 98% of the methyl groups from O6-methylguanine present in alkylated DNA, but leaves N-alkylated purines untouched. Single-stranded DNA containing O6-methylguanine is a poor substrate, with the methyl transfer occurring at approximately 0.1% of the rate for duplex DNA. This latter observation may explain the high frequency of mutations induced by alkylating agents at DNA replication forks.