ANALYSIS OF ACYL-COENZYME A BINDING TO THE TRANSCRIPTION FACTOR FADR AND IDENTIFICATION OF AMINO-ACID-RESIDUES IN THE CARBOXYL-TERMINUS REQUIRED FOR LIGAND BINGING

The Escherichia coil FadR protein regulates the transcription of many unlinked genes and operons encoding proteins required for fatty acid s ynthesis and degradation. Previously, we demonstrated that the ability of purified FadR to bind DNA in vitro is inhibited by long chain acyl coenzyme A esters (DiRusso, D. D., Heimert, T. L., and Metzger, A, K. (1992) J. Biol. Chem. 267, 8685-8691). In the present work, we show t hat FadR binds acyl-CoA directly. Ligand binding resulted in a shift i n the apparent pI of FadR from 6.9 to 6.2 and in a marked decrease in intrinsic fluorescence. The K-m for FadR binding of oleoyl coenzyme A was determined to be 12.1 nM using the fluorescence quenching assay, T he binding site for acyl-CoA was identified by selection of noninducib le mutations in the FadR gene. One altered protein carrying the change Ser(219) to Asn (S219N) was purified and shown to have a reduced affi nity for oleoyl coenzyme A as evidenced by a K-m of 257 nM. S219N reta ined the ability to bind DNA and to repress or activate transcription. Alanine substitution of amino acid residues 215 through 230 identifie d Gly(216) and Trp(223) as also required specifically for induction. T his region of FadR shares amino acid identities and similarities with the coenzyme A-binding site of Clostridium thermoaceticum CO dehydroge nase/acetyl-coenzyme A synthase. Due to the alteration in binding affi nity of the purified S219N protein, the non-inducible phenotype of sev eral proteins carrying alanine substitutions and similarities to CO de hydrogenase/acetyl-coenzyme A synthase we propose this region of FadR forms part of the acyl-CoA-binding domain.