Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

R J Whitaker,M J Fiske,R A Jensen

doi:10.1016/s0021-9258(18)33582-8

Abstract

In Pseudomonas aeruginosa the initial enzyme of aromatic amino acid biosynthesis, 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase, has been known to be subject to feedback inhibition by a metabolite in each of the three major pathway branchlets. Thus, an apparent balanced multieffector control is mediated by L-tyrosine, by L-tryptophan, and phenylpyruvate. We have now resolved DAHP synthase into two distinctive regulatory isozymes, herein denoted DAHP synthase-tyr (Mr = 137,000) and DAHP synthase-trp (Mr = 175,000). DAHP synthase-tyr comprises greater than 90% of the total activity. L-Tyrosine was found to be a potent effector, inhibiting competitively with respect to both phosphoenolpyruvate (Ki = 23 microM) and erythrose 4-phosphate (Ki = 23 microM). Phenylpyruvate was a less effective competitive inhibitor: phosphoenolpyruvate (Ki = 2.55 mM) and erythrose 4-phosphate (Ki = 1.35 mM). DAHP synthase-trp was found to be inhibited noncompetitively by L-tryptophan with respect to phosphoenolpyruvate (Ki = 40 microM) and competitively with respect to erythrose 4-phosphate (Ki = 5 microM). Chorismate was a relatively weak competitive inhibitor: phosphoenolpyruvate (Ki = 1.35 mM) and erythrose 4-phosphate (Ki = 2.25 mM). Thus, each isozyme is strongly inhibited by an amino acid end product and weakly inhibited by an intermediary metabolite.

Highlights

Heptulosonate 7-phosphate (DAHP)synthase, hasbeen These include isozymic feedback inhibition, sequential feedknown to be subjectto feedback inhibition by a metab- back inhibition, cumulative feedback inhibition,and concerted olite in each of the three major pathway branchlets
Since 1973 (5) the DAHP synthase activoitfyP. aeruginosa has been knownto be feedbackinhibited by L-tryptophan and by phenylpyruvate in addition to theearlier known (4)feedback inhibition by L-tyrosine
This patterncomprised anovel, but apparently balanced allosteric pattern, in which a single metabolite within each terminal branchlet was an allosteric effector of DAHP synthase

Summary

MATERIALS AND METHODS

Chorismate was a relatively weak competitive inhibi- Microbiological Aspects-Strain 1 of P.aeruginosa, originally tor: phosphoenolpyruvate(Ki= 1.35 mM) and erythrose obtainedfrom B. Theneed for in-depth studiesis illustrated by thefactthatDAHPsynthase of Pseudomonas aeruginosa was originally classified as sucha unimetabolitecontrolled enzyme (4).In cases where exogenous amino acids according to the method of Calhoun et al (11).In crude extracts, as well aspartially purified samples of each isozyme, conditions of proportionality with respect to both time and protein concentration were observed. The costs of publication of this sulfate prepared in 50 mM potassium phosphatebuffer (pH 7.0) were article were defrayed in part by the payment of page charges This added to reaction mixtures t o a final concentration of 1.0 mM to test article must be hereby marked “aduertisement” in accord- for optimal activity in each enzyme sample (crude extract and paranc’ eThweitahb1b8reUv.iSa.tCio.nSuescetidoins:1D73A4HsoPl,e3ly-dtoeoixnyd-i~ca-taerathbiisnofa-chte.ptuloson-ttirapl)ly.Tphueraifpiepdrosparmiaptelems of eta DAHP l ion synthase-tyr and DAHP synthasewas included in subsequent ate 7-phosphate. While each of the partially purified isozymes was activated best by cobalt, crude extracts were activated best by manganese

RESULTS

Crude extract

Findings

Ibl i