William V. Shaw

Chloramphenicol Acetyltransferase: Enzymology and Molecular Biology

Naturally occurring chloramphenicol resistance in bacteria is normally due to the presence of the antibiotic inactivating enzyme chloramphenicol acetyltransferase (CAT) which catalyzes the acetyl-S-CoA-dependent acetylation of chloramphenicol at the 3-hydroxyl group. The product 3-acetoxy chloramphenicol does not bind to bacterial ribosomes and is not an inhibitor of peptidyltransferase. The synthesis of CAT is constitutive in E. coli and other Gram-negative bacteria which harbor plasmids bearing the structural gene for the enzyme, whereas Gram-positive bacteria such as staphylococci and streptococci synthesize CAT only in the presence of chloramphenicol and related compounds, especially those with the same stereochemistry of the parent compound and which lack antibiotic activity and a site of acetylation (3-deoxychloramphenicol). Studies of the primary structures of CAT variants suggest a marked degree of heterogeneity but conservation of amino acid sequence at and near the putative active site. All CAT variants are tetramers composed in each case of identical polypeptide subunits consisting of approximately 220 amino acids. The catalytic mechanism does not appear to involve an acyl-enzyme intermediate although one or more cysteine residues are protected from thiol reeagents by substrates. A highly reactive histidine residue has been implicated in the catalytic mechanism.

The amino acid sequence of the delta haemolysin of Staphylococcus aureus

The 6 haemolysin of Sfaphylococcus aureus is one of a number of extracellular cytolytic and cytotoxic polypeptides produced by most strains of the microorganism [l-4] and is notable for its physlco-chemical and biological properties. It is stable to boiling and soluble in chloroform-methanol (2: 1, v/v) and aqueous solutions (reviewed [4]) and is inhibited by phospholipids, fatty acids, and normal serum lipoproteins [5-71. Three different purification procedures [8-lo] yield material with broadly similar properties and an amino acid analysis which is notable for the absence of arginine, proline, histidine, tyrosine and cysteine, and the presence of a high proportion of hydrophobic amino acids. Although most earlier studies suggested that native 6 haemolysin was likely to have mol. wt >lOO 000, it was apparent from gel permeation chromatography under denaturing conditions that the minimum molecular weight for the polypeptide subunit was likely to be <6000 [lo]. This investigation of the primary structure of 6 haemolysin was prompted by the realization that the solvent-transfer method 181 provided substantial quantities of highly purified 6 haemolysin from culture filtrates of S. aureus, that it did so more rapidly and reproducibly than other methods [7,8] and that the polypeptide shared all of the characteristic amphipathic and cytolytic properties observed with preparations of 6 haemolysin previously purified by ion exchange or adsorption chromatography and by gel filtration. The most striking properties of 6 haemolysin are

PURIFICATION AND CHARACTERIZATION OF PHOSPHOPANTETHEINE ADENYLYLTRANSFERASE FROM ESCHERICHIA COLI

Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis: the reversible adenylation of 4′-phosphopantetheine yielding 3′-dephospho-CoA and pyrophosphate. Wild-type PPAT fromEscherichia coli was purified to homogeneity. N-terminal sequence analysis revealed that the enzyme is encoded by a gene designated kdtB, purported to encode a protein involved in lipopolysaccharide core biosynthesis. The gene, here renamedcoaD, is found in a wide range of microorganisms, indicating that it plays a key role in the synthesis of 3′-dephospho-CoA. Overexpression of coaD yielded highly purified recombinant PPAT, which is a homohexamer of 108 kDa. Not less than 50% of the purified enzyme was found to be associated with CoA, and a method was developed for its removal. A steady state kinetic analysis of the reverse reaction revealed that the mechanism of PPAT involves a ternary complex of enzyme and substrates. Since purified PPAT lacks dephospho-CoA kinase activity, the two final steps of CoA biosynthesis in E. coli must be catalyzed by separate enzymes.

Structural and Mechanistic Studies of Galactoside Acetyltransferase, the Escherichia coli LacA Gene Product

Escherichia coli galactoside acetyltransferase (GAT) is a member of a large family of acetyltransferases that O-acetylate dissimilar substrates but share limited sequence homology. Steady-state kinetic analysis of overexpressed GAT demonstrated that it accepted a range of substrates, including glucosides and lactosides which were acetylated at rates comparable to galactosides. GAT was shown to be a trimeric acetyltransferase by cross-linking with dimethyl suberimidate. Fluorometric analysis of coenzyme A binding showed that there is a fluorescence quench associated with acetyl-CoA binding whereas CoA has no effect. This difference was exploited to measure dissociation rates for both CoA and acetyl-CoA by stopped-flow fluorometry. The rate of dissociation of CoA (2500 s−1) is at least 170-fold faster than kcat for any substrate tested. The fluorescence response to acetyl-CoA binding is entirely due to Trp-139 since replacement by phenylalanine completely abolished the fluorescence quench. Treatment of GAT by [14C]iodoacetamide resulted in complete inactivation of the enzyme and the incorporation of label into histidyl and cysteinyl residues to approximately equal extents. Following replacement of His-115 by alanine, label was incorporated solely into cysteinyl residues. Furthermore, the substitution results in an 1800-fold decrease in kcat suggesting that His-115 has an important catalytic role in GAT.

Chloramphenicol Acetyltransferases Determined by R Plasmids from Gram-negative Bacteria

Summary: The mechanism of resistance to chloramphenicol specified by 18 plasmids from Gram-negative bacteria representing different incompatibility groups was investigated. Most determined the drug-inactivating enzyme chloramphenicol acetyltransferase. The enzymes were purified and their properties were compared with those of the previously characterized enzyme types specified by R429 (type I), s-a (type II) and R387 (type III). The type I enzyme was determined by plasmids representing incompatibility groups FII, C, S, I, H, L, O and Com9. Plasmids from incompatibility groups K, Iγ and the A-C complex specified the type III enzyme, while elements representing incompatibility groups V and W determined the type II variant.

Analysis of two chloramphenicol resistance plasmids from Staphylococcus aureus: Insertional inactivation of Cm resistance, mapping of restriction sites, and construction of cloning vehicles ☆

Abstract Chloramphenicol (Cm) resistance plasmids pCW7 and pC221 of Staphylococcus aureus have been characterized by the construction of detailed restriction maps and by the identification of restriction sites on both plasmids which map within either the structural gene encoding CAT or its controlling elements. The number and order of recognition sites for endonucleases AluI, HinfI, and TaqI on pCW7 and pC221 were determined from multiple enzyme digestion results and by the 5′-terminal labeling procedure of Smith and Birnstiel (1976) . Endonuclease sites mapping within the Cm-resistance determinant were identified by the construction of recombinant plasmids in vitro from pC221 or pCW7 and the tetracycline-resistance plasmid pCW3. Site-specific mutations were then introduced by filling in the complementary ends of selected restriction sites present on pCW7 or pC221 with E. coli polymerase I followed by recircularization of the recombinant plasmid by blunt-end ligation. Pol I treatment of the BstEII site of pC221 and the BstEII or BglII site present on pCW7 resulted not only in the loss of those recognition sites but also in the loss of Cm resistance encoded by the plasmid. Circularization of the largest MboI fragment (1.8kb) from pC221 formed a stable replicon which encoded an inducible CAT but did not exhibit the relaxation phenomenon associated with pC221. The possible roles of several of the recombinant plasmids as cloning vehicles within S. aureus and Bacillus subtilis are discussed.

Inactivation of Chloramphenicol by O-Phosphorylation A NOVEL RESISTANCE MECHANISM IN STREPTOMYCES VENEZUELAE ISP5230, A CHLORAMPHENICOL PRODUCER

Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic DNA from the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, confers resistance when introduced by transformation into the Cm-sensitive host Streptomyces lividans M252 (Mosher, R. H. Ranade, N. P., Schrempf, H., and Vining, L. C.(1990) J. Gen. Microbiol. 136, 293-301). Transformants rapidly metabolized Cm to one major product, which was isolated and purified by reversed phase chromatography. The metabolite was identified by nuclear magnetic resonance spectroscopy and mass spectrometry as 3′-O-phospho-Cm, and was shown to have negligible inhibitory activity against Cm-sensitive Micrococcus luteus. The nucleotide sequence of the S. venezuelae DNA insert in pJV4 contains an open reading frame (ORF) that encodes a polypeptide (19 kDa) with a consensus motif at its NH terminus corresponding to a nucleotide-binding amino acid sequence (motif A or P-loop; Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J.(1982) EMBO J. 1, 945-951). When a recombinant vector containing this ORF as a 1.6-kilobase pair SmaI-SmaI fragment was used to transform S. lividans M252, uniformly Cm-resistant transformants were obtained. A strain of S. lividans transformed by a vector in which the ORF had been disrupted by an internal deletion yielded clones that were unable to phosphorylate Cm, and exhibited normal susceptibility to the antibiotic. The results implicate the product of the ORF from S. venezuelae as an enzymic effector of Cm resistance in the producing organism by 3′-O-phosphorylation. We suggest the trivial name chloramphenicol 3′-O-phosphotransferase for the enzyme.

Affinity and hydrophobic chromatography of three variants of chloramphenicol acetyltransferases specified by R factors in Escherichia coli

The high level resistance of E. coli to chloramphenicol (CM) is the result of antibiotic acetylation by the enzyme chloramphenicol acetyltransferase (EC 2.3.1.99) [ 1,2 ] . Three distinguishable types of chloramphenicol acetyltransferase (CAT) have been found in E. coli strains bearing an episomal (R factor) determinant for chloramphenicol resistance [3,4]. Two out of the three variants have been found to be associated with so-called fiR factors and one with fi’ factors. Each variant is composed of four identical subunits of approx. 20 000 mol. wt. [5]. Differences in electrophoretic mobility, inhibition by sulfhydryl reagents, K, for chloramphenicol, and immunological behaviour have been observed [4]. Although purification of the CAT variants by conventional methods has been successful [6,7], their use has resulted in variable overall yield, especially when purification on a large scale has been attempted. In this report we describe the use of the technique of affinity chromatography [8] to achieve purification to homogeneity of CAT in high yield. The approach taken was to attach the free amino of chloramphenicol (CM base) or of p-amino chloramphenicol (fig.2) to the free carboxyl group of substituted Sepharose [Seph-NH-(CH&-COOH] by formation of an amide bond following activation by a water soluble carbodiimide.

Chloramphenicol Acetyltransferases Specified by fi− R Factors

Chloramphenicol acetyltransferases specified by fi− (F fertility-noninhibiting) R factors were compared with those specified by the fi+ R factors R1, 222, and R6-S. Three classes of enzyme were distinguished: class I was determined by group N fi− R factors and was indistinguishable from the enzyme determined by fi+ R factors, class II was correlated with group W fi− R factors, and class III was determined by R387, an unclassified fi− R factor. Images

The serine acetyltransferase from Escherichia coli: Over-expression, purification and preliminary crystallographic analysis

An expression vector has been constructed which increases the expression of serine acetyltransferase (SAT) from E. coli to 17% of the soluble cell protein. A novel purification procedure, using dye‐affinity chromatography, allows purification of SAT to homogeneity. The enzyme has been crystallised from polyethylene glycol, in the presence of L‐cysteine (an inhibitor of SAT). The crystals which diffract to beyond 3.0 Å resolution are of the tetragonal spacegroup P41212(or P43212) with cell dimensions a = b = 123 Å, c = 79 Å. Since ultracentrifugation and gel‐filtration experiments indicate that purified SAT is a tetramer, there appears to be one‐half tetramer in the asymmetric unit (V m = 2.55 Å3/Da).