Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing

Abstract

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.

FIG. 1

Enzymes with mutations in the N-terminal nucleophile and residues for the oxyanion hole are not processed. The wild-type enzyme from plasmid pTrcBFc (lane 1) and enzymes with mutations C1T (lane 2), G103A (lane 3), and N102D (lane 4) were resolved by electrophoresis on an SDS–8% polyacrylamide gel. Arrows identify the proenzyme and mature enzyme species.

FIG. 2

Processing of PRTase flexible-loop-deleted glutamine PRPP amidotransferase. The PRTase flexible-loop-deleted enzyme (lane 1) and an H70N enzyme (lane 2) as a size marker were resolved by electrophoresis on an SDS–8% polyacrylamide gel. Arrows identify the proenzyme and mature enzyme species.

FIG. 3

Effect on processing of mutations in conserved histidine residues in the glutaminase domain and in conserved glutamates in the propeptide. Enzymes with mutations in H25Q (lane 1), H70Y (lane 2), H70N (lane 3), H101Q (lane 4), E2G (lane 5), E-2G/E-1A (lane 6), and the wild type (lane 7) were resolved by electrophoresis on an SDS–8% polyacrylamide gel. Arrows identify the proenzyme and mature enzyme species.

FIG. 4

No processing of mutant N102D subunits by wild-type subunits in a hetero-oligomer. In experiment 1 (lanes 1 to 3) hetero-oligomers were made in vivo from plasmid pTrc(N102D)c-wt encoding N102D proenzyme with a His tag and proenzyme wild type (no His tag). Lane 1, extract; lane 2, proteins purified by using the His tag under native conditions; lane 3, proteins purified by using the His tag under denaturing conditions. In experiment 2 (lanes 4 to 6) heterooligomers were made in vivo from plasmid pTrc(N102D)c-Δwt encoding N102D proenzyme with a His tag and mature wild-type enzyme. Samples in lanes 4 to 6 correspond to the samples in lanes 1 to 3. Proteins were resolved by electrophoresis on an SDS–8% polyacrylamide gel. Arrows mark the positions of the proenzyme and mature enzyme species.