An NADH-Dependent Acetoacetyl-CoA Reductase from Euglena gracilis: Purification and Characterization, Including Inhibition by Acyl Carrier Protein

An NADH-dependent acetoacetyl-CoA reductase from Euglena gracilis variety bacillaris was extensively purified and characterized. Two different isoelectric forms of the reductase with identical characteristics otherwise were found. The reductase was noncompetitively inhibited by acyl carrier protein, K(i) 5.6 micromolar at pH 5.4; this inhibition decreased with increasing pH or ionic strength. Coenzyme A was a competitive inhibitor, K(i) 230 micromolar. Kinetic parameters with respect to acetoacetyl-CoA and NADH were sensitive to changes in pH and ionic strength

Hemolysin Toxin Activation, an Internal Fatty Acylation

Escherichia coli hemolysin (HlyA) is synthesized as nontoxic proHlyA. It is post-translationally modified by the internal protein transacylase HlyC to form toxic HtyA. The enzyme transfers a long chain fatty acid from acyl-acyl carrier protein (acyl-ACP) to one or two internal lysine residues forming an amide bond. HlyA is secreted and forms cation-specific channels in target cells leading to osmotic lysis. Acylation is the single factor that renders HlyA toxic. Previously erythrocyte hemolysis was used to monitor the function of the enzyme. A direct assay was developed in our laboratory using acyl-ACPs with radiolabelled acyl groups which permitted quantification of transacylase activity. Examination of diverse acyl-ACPs revealed that myristoyl-ACP was the preferred fatty acyt donor. CoA esters did not serve as substrates, ACPSIt (non-acylated ACP) was inhibitory. The different acyl-toxins generated in the direct assay were monitored for erythrocyte lysis, and the efficacy of the various acyl-ACPs as substrates differed from the lytic abilities of the corresponding acyl-toxins that were produced. Notably, an aeyl-enzyme intermediate was detected and characterized. Denaturing polyacrylamide gel electrophoresis of the acyl-enzyme intermediate, size exclusion chromatography, and chemical cross linking experiments between HlyC and acyl-ACP all indicated that the active form of the enzyme was a monomer. HlyC does not resemble any other known transacylase, and some confusion exists regarding its role in the fatty acylation of proHlyA. We have purified HlyC and demonstrated that the pure protein has transacylase activity; thus HlyC is the enzyme

Insights Into the Catalytic Mechanism of HlyC, the Internal Protein Acyltransferase That Activates Escherichia Coli Hemolysin Toxin

Hemolysin, a toxic protein secreted by pathogenic Escherichia coli, is converted from nontoxic prohemolysin, proHlyA, to toxic hemolysin, HlyA, by an internal protein acyltransferase, HlyC. Acylacyl carrier protein (ACP) is the essential acyl donor. The acyltransferase reaction proceeds through two partial reactions and entails formation of a reactive acyl- HlyC intermediate [Trent, M. S., Worsham, L. M., and Ernst-Fonberg, M. L. (1999) Biochemistry 38, 9541-9548]. The ping pong kinetic mechanism implied by these findings was validated using two different acyl-ACP substrates, thus verifying the independence of the previously demonstrated two partial reactions. Assessments of the stability of the acyl-HlyC intermediate revealed an increased stability at pH 8.6 compared to more acidic pHs. Mutations of a single conserved histidine residue essential for catalysis gave minimal activity when substituted with a tyrosine residue and no activity with a lysine residue. Unlike numerous other His23 mutants, however, the H23K enzyme showed significant acyl-HlyC formation although it was unable to transfer the acyl group from the proposed amide bond intermediate to proHlyA. These findings are compatible with transient formation of acyl-His23 during the course of HlyC catalysis. The effects of several other single site-directed mutations of conserved residues of HlyC on different portions of the reaction progress were examined using a 39 500 kDa fragment of proHlyA which was a more effective substrate than intact proHlyA