The Escherichia coli gusA Gene

Originally, p-glucuronidase was biochemically characterized in the bacterium Escherichia coli14 and later the gusA gene was isolated from E. coli strain K12.15 GUS activity enables E. coli in its natural environment, the gut, to decouple glucuronic acid from various substrates13 and to use glucuronic acid for further metabolization. The compounds coupled to glucuronic acid, the aglycones, can be very diverse. They are coupled to glucuronic acid in the liver to make them more water-soluble and are taken up again after decoupling by E. coli. This circulation is caled the enterohepathic cycle, an important physiological process in the human body. In E. coli, gusA is part of an operon, together with two other genes, gusB and gusC, encoding a glucuronide-specific permease and a membrane-associated protein of unknown function, respectively (Fig. 6.1). The E. coli gusA gene is 1809 bp long and the p-glucuronidase has a predicted monomi molecular weight of 68,300, in agreement with the experimentally determined molecular weight of 73,000.15 The enzyme is probably active as a tetramer in vivo.11 Genetic analysis of the gusA locus has revealed three distinct modes of transcriptional control in E. coli. Firstly, the GusR repressor binds to an operator sequence upstream of gusA and is relieved from binding in the presence of glucuronide substrates. A second repressor, UxuR, downregulates both the gusA gene and the uxuAB operon,16,17 required for further metabolization of the glucuronic acid. A third level of control is exerted by catabolite repression15

The p-glucuronidase of E. coli specifically hydrolyzes p-linked D-glucuronides to D-glucuronic acid and aglycone. The enzyme has a broad specificity range for p-conjugated glucuronides but not for other glycosides18 p-glucuronidase was found to be very stable and to be more active in the presence of thiol-reducing agents such as p-mercaptoethanol and dithiothreitol (DTT).11 The optimum pH for GUS activity is between 5.0 and 6.8, but the enzyme retains about 50% activity at pH 4.3 or at pH 8.4. GUS is partially resistant to

Coli Internal
Table 6.1. Characteristics of Escherichia coli p-glucuronidase (see text for ref.)

Characteristics

Description

Substrates

p-linked D-glucuronides are hydrolized to D-glucuronic acid

and aglycone

Exo-hydrolase

No cleavage of glucuronides in internal positions within

polymers (plant cell wall)

Molecular weight

Theoretical 68,000 ; experimental 72,000

pH range

5.0-7.0 (activity is 50% at pH 4.3 and 8.5)

Cofactors

None

Thermal stability

Half-life of two hours at 55°C and of 15 minutes at 60°C

Special requirements

Enhanced activity in presence of thiol reducing agents (DTT)

thermal inactivation with a half-life of two hours at 55°C and about 15 minutes at 60°C. GUS is inhibited by some heavy divalent metal ions such as Cu2+ and Zn2+.11 Physical and enzymatic characteristics of GUS are summarized in Table 6.1.

The enzyme can tolerate large amino-terminal additions for translational fusions. It is not processed at the amino-terminus in E. coli and is found exclusively in the cytoplasm. It is not subject to any post-transcriptional modification.

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