Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105

Abstract

Penicillin G acylase from Escherichia coli ATCC 11105 is synthesized from its precursor polypeptide into a catalytically active heterodimer via a complex posttranslational processing pathway. Substitutions in the pair of aminoacyl residues at the cleavage site for processing the small and large subunits were made. Their processing phenotypes and penicillin G acylase activities were analyzed. By the introduction of a prolyl residue at either position, the processing of the small subunit was blocked without a change in enzymatic activity. Four other substitutions had no effect. At the site for processing the large subunit, four substitutions out of the seven examined blocked processing. In general, penicillin G acylase activity seemed to be proportional to the efficiency of the large-subunit-processing step. Ser-290 is an amino acid critical for processing and also for the enzymatic activity of penicillin G acylase. In the mutant pAATC, in which Ser-290 is mutated to Cys, the precursor is processed, but there is no detectable enzymatic activity. This suggests that there is a difference in the structural requirements for the processing pathway and for enzymatic activity. Recombination analysis of several mutants demonstrated that the small subunit can be processed only when the large subunit is processed first. Some site-directed mutants from which signal peptides were removed showed partial processing phenotypes and reduced enzymatic activities. Their expression showed that the prerequisite for penicillin G acylase activity is the efficient processing of the large subunit and that the maturation of the small subunit does not affect the enzymatic activity.Penicillin G acylase (PGA) is an enzyme that catalyzes the hydrolysis of benzylpenicillin to give 6-aminopenicillanic acid, an intermediate in the production of semisynthetic penicillin (28). PGA from Escherichia coli ATCC 11105 is a periplasmic enzyme that is composed of the small subunit and the large subunit. For PGA to reach its catalytically active dimer form in the periplasm, multistep proteolytic processing from a single polypeptide must be accomplished by removing a signal peptide (26 residues) and a spacer peptide (54 residues) (24, 25). The proteolytic cleavage sites are located between the Ala-235-Ala-236 and Thr-289-Ser-290 pairs (18). This maturation mechanism is peculiar to PGA as a bacterial enzyme, though many viral proteins and eucaryotic polypeptide hormones require activation by proteolytic cleavage processes (6,7). But the enzymes involved in each processing step and their locations within the cell have not yet been identified.In this report, site-specific mutations at the proteolytic cleavage sites of PGA were constructed in order to obtain the necessary information for cleavage activation, especially (i) the amino acids critical to PGA processing and PGA activity, (ii) the amino acid specificity of each cleavage step, and (iii) the order of the cleavage steps. Through the expression of various recombinantpga genes, the characteristics of each...

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

“…One of the conformations revealed that Ser264 in the active site is positioned such that its Og group could attack the appropriate carbonyl carbon atom of the scissile bond. Moreover, this active site Ser264 is absolutely essential for activity (Choi et al 1992). Strikingly, from the structure, there was no obvious candidate for the general base in the proteolytic reaction (Hewitt et al 2000).…”

Why Does Threonine, and Not Serine, Function as the Active Site Nucleophile in Proteasomes?

“…These enzymes, therefore, have been termed N-terminal nucleophile (Ntn) hydrolases (19). This N-terminal residue is cysteine in glutamine-5-phosphoribose-1-pyrophosphate amidotransferase (20), glucosamine-6-phosphate synthase (21), and asparagine synthase (22); serine in penicillin acylase (23,24); and threonine in glycosylasparaginase (25,26), all proteasomes, and their bacterial homologue HslVU (27). A variety of observations indicate that proteasomes indeed cleave peptide bonds by this unusual mechanism, in which a hydroxyl group of the N-terminal threonine serves as the catalytic nucleophile: (i) replacement of this threonine by an alanine in archaeal (28) and yeast (29 -32) proteasomes abolishes their proteolytic activity; (ii) the hydroxyl group of this threonine is modified by irreversible inhibitors lactacystin (33,34), 3,4-dichloroisocoumarin (10,35), and vinyl sulfone (36); and (iii) it has been shown by x-ray diffraction to form a hemiacetal bond with the peptide aldehyde inhibitor (9,34).…”

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

“…It was revealed that both Thr 263 and Ser 264 were crucial for the first cleavage of PGA. Substitution of Ser 264 with Thr, Arg, or Gly resulted in the complete failure of enzyme activation (39). Substitution of Thr 263 with Ser or Cys had less effect on the first cleavage but substitution with Gly also resulted in the failure of the normal first cleavage between Thr 263 and Ser 264 .…”

“…Specific Binding and Covalent Binding for the Second Cleavage-In the previous studies, besides CA, PGA and PvdQ were confirmed to be Ntn hydrolases that require two-step proteolysis for activation, releasing spacers consisting of 54, 22, or 23 aa, respectively (39,40) (Table 4). The long distances between the second cleavage sites and the Ntn Ser residues were also observed in the respective crystal structures (7,41).…”