THE AROQ-ENCODED MONOFUNCTIONAL CHORISMATE MUTASE (CM-F) PROTEIN IS APERIPLASMIC ENZYME IN ERWINIA-HERBICOLA

Enteric bacteria possess two species of chorismate mutase which exist as catalytic domains on the amino termini of the bifunctional PheA and TyrA proteins. In addition, some of these organisms possess a third c horismate mutase, CM-F, which exists as a small monofunctional protein . The CM-F gene (denoted aroQ) from Erwinia herbicola was cloned and s equenced for the first time. A strategy for selection by functional co mplementation in a chorismate mutase-free Escherichia coli background was devised by using a recombinant plasmid derivative of pUC18 carryin g a Zymomonas mobilis tyrC insert which encodes cyclohexadienyl dehydr ogenase. The aroQ gene is 543 bp in length, predicting a 181-residue p rotein product having a calculated molecular mass of 20,299 Da. The E. herbicola aroQ promoter is recognized by E. coli, and a putative sigm a-70 promoter region was identified. N-terminal amino acid sequencing of the purified CM-F protein indicated cleavage of a 20-residue signal peptide. This was consistent with the monomeric molecular mass determ ined for the enzyme of about 18,000 Da. The native enzyme is a homodim er. The implied translocation of CM-F was confirmed by osmotic shock e xperiments which demonstrated a periplasmic location. Immunogold elect ron microscopy indicated a polar localization within the periplasm. Po lyclonal antibody raised against E. herbicola CM-F did not cross-react with the CM-F protein from the closely related Serratia rubidaea, as well as from a number of other gram-negative bacteria. Furthermore, wh en the E. herbicola aroQ gene was used as a probe in Southern blot hyb ridizations with EcoRI digests of chromosomal DNA from S. rubidaea and other enteric organisms, no hybridization was detected at low stringe ncy. Thus, the aroQ gene appears to be unusually divergent among close ly related organisms. The deduced CM-F amino acid sequence did not exh ibit compelling evidence for homology with the monofunctional chorisma te mutase protein of Bacillus subtilis.