Characterization and comparison of chloramphenicol acetyltransferase variants.

1. Variants of chloramphenicol acetyltransferase from a variety of bacterial species have been isolated and purified to homogeneity. They constitute a heterogeneous group of proteins as judged by analytical affinity and hydrophobic ('detergent') chromatography, native and sodium dodecyl sulfate electrophoresis, sensitivity to sulfhydryl specific reagents, steady state kinetic analysis, and reaction with antisera. 2. The most striking observation is that three variants of chloramphenicol acetyltransferase (R factor type III, Streptomyces acrimycini, and Agrobacterium tumefaciens) possess an apparent subunit molecular weight (24,500) which is significantly greater than that of all other variants examined (22,500). The three atypical variants are not identical since they show marked differences in a number of important parameters. 3. Although the fundamental mechanism of catalysis may prove to be identical for all chloramphenicol acetyltransferase variants, there is a wide range of sensitivity to thiol-directed inhibitors among the enzymes studied. 4. Amino acid sequence analysis of the N-termini of selected variants suggests that the qualitative differences among chloramphenicol acetyltransferase variants is a reflection of structural heterogeneity which is most marked in comparisons between variants from Gram-positive and Gram-negative species.     

Expression of two recombinant chloramphenicol acetyltransferase variants in highly reduced genome Escherichia coli strains

Highly reduced E. coli strains, MDS40, MDS41, and MDS42, lacking approximately 15% of the genome, were grown to high cell densities to test their ability to produce a recombinant protein with high yields. These strains lack all transposons and insertion sequences, cryptic prophage and many genes of unknown function. In addition to improving genetic stability, these deletions may reduce the biosynthetic requirements of the cell potentially allowing more efficient production of recombinant protein. Basic growth parameters and the ability of the strains to produce chloramphenicol acetyltransferase (CAT) under high cell density, batch cultivation were assessed. Although growth rate and recombinant protein production of the reduced genome strains are comparable to the parental MG1655 strain, the reduced genome strains were found to accumulate significant amounts of acetate in the medium at the expense of additional biomass. A number of hypotheses were examined to explain the accumulation of acetate, including oxygen limitation, carbon flux imbalance, and metabolic activity of the recombinant protein. Use of a non-catalytic CAT variant identified the recombinant protein activity as the source of this phenomenon; implications for the metabolic efficiency of the reduced genome strains are discussed.     

Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase

Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria.     

Comparison of chloramphenicol acetyltransferase variants in staphylococci. Purification, inhibitor studies and N-terminal sequences.

Four electrophoretic variants of chloramphenicol acetyltransferase (types A, B, C and D) found in chloramphenicol-resistant staphylococci were purified by affinity chromatography. Michaelis constants and the kinetics of inactivation with a variety of reagents for the four variants are virtually identical. Their similar amino acid compositions and near identical N-terminal sequences suggest a high degree of overall sequence homology. The thiol-specific reagents 5,5'-dithiobis-(2-nitrobenzoic acid), 2-nitro-5-thiocyanobenzoic acid and 2,2'-dithiopyridine are without significant effect on enzyme activity, whereas 1-fluoro-2,4-dinitrobenzene, N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and, particularly, bromoacetyl-CoA and diethyl pyrocarbonate are potent inhibitors. Iodoacetate is not an inhibitor. The results of chemical modification studies on the four enzyme variants and the identification of 3-carboxymethylhistidine in acid hydrolysates of one variant (type C) after inactivation with iodoacetamide suggest that a unique histidine residue may be involved in the mechanism of catalysis.     

The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts.

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.     

Pseudosecretion of Escherichia coli chloramphenicol acetyltransferase by Bacillus subtilis.

Bacillus subtilis harboring the vector 25RBSII secrets an Escherichia coli-derived chloramphenicol acetyltransferase into culture supernatants. The secreted enzyme lacks 18 amino acids; these are removed externally rather than during secretion.     

Nonlinear steady-state kinetics of chloramphenicol acetyltransferase.

Steady-state kinetic analysis of chloramphenicol acetyltransferase showed that medium effects (higher temperatures or pH, higher ionic strengths, or lower values for dielectric constant) altered the kinetic behaviour of the enzyme with acetyl-CoA as substrate, but did not significantly affect behaviour with chloramphenicol. This was manifest as an increase in the degree of the rate equation to a 2:2 function. This is interpreted in terms of perturbations to the enzyme at or near the acetyl-CoA binding region of the enzyme.     

Characterization of the chloramphenicol acetyltransferase variants encoded by the plasmids pSCS6 and pSCS7 from Staphylococcus aureus.

The two 4.6 kb chloramphenicol resistance (CmR) plasmids pSCS6 and pSCS7, previously identified in Staphylococcus aureus from subclinical bovine mastitis, both encoded an inducible chloramphenicol acetyltransferase (CAT, EC 2.3.1.28). The pSCS6- and pSCS7-encoded CAT variants were purified by ammonium sulphate precipitation, ion-exchange chromatography and fast protein liquid chromatography (FPLC). Both native enzymes showed Mr values of 70,000 on FPLC and were composed of three identical subunits, each of Mr approximately 23,000. The CAT variants from pSCS6 and pSCS7 differed in their net charges and in their isoelectric points. The isoelectric point of the CAT from pSCS6 was pH 5.7 and that of the CAT from pSCS7 pH 5.2. Both CAT variants exhibited highest enzyme activities at pH 8.0. The Km values for chloramphenicol and acetyl-CoA of the CAT from pSCS6 were 2.5 microM and 58.8 microM, respectively, while those of the CAT from pSCS7 were 2.7 microM and 55.5 microM. Both CAT variants were relatively thermostable. The CAT from pSCS6 was less sensitive to mercuric ions than the CAT from pSCS7.     

Ligand interaction energies and molecular recognition by chloramphenicol acetyltransferase.

The apparent binding energy for the interaction of the 3-hydroxyl group of chloramphenicol (CM) with the proposed general base (His-195) in chloramphenicol acetyltransferase (CAT) was determined by comparison of the dissociation constants of CM and 3-deoxyCM with CAT. The delta Gapp for this hydrogen bond to the N-3 of the imidazole ring is 1.5 kcal mol-1. Extending the use of modified ligands, in an approach which is complementary to that of site-directed mutagenesis, the binding affinity of each of a family of 3-halo-3-deoxychloramphenicol derivatives was observed to increase in the series F less than Cl less than Br less than I and is dominated by hydrophobic considerations. There is a linear free energy relationship between the dissociation constants for binding to CAT and an empirical hydrophobicity scale derived from reverse-phase HPLC retention times. The existence of such a relationship allows a true estimate of the total energetic contribution of interactions between the 3-hydroxyl group of CM and its contacts at the active site of CAT to be made on the basis of a regression analysis. The calculated value of delta Gbind (2.7 kcal mol-1) must include not only the hydrogen bond but also some favorable van der Waals interactions. The results demonstrate some of the advantages of an analysis of the energetics of ligand binding using modified ligands, in an approach that is formally analogous with and complementary to the use of site-directed mutations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complementation of buried lysine and surface polar residues in a designed heterodimeric coiled coil

The coiled coil is an attractive target for protein design. The helices of coiled coils are characterized by a heptad repeat of residues denoted a to g. Residues at positions a and d form the interhelical interface and are usually hydrophobic. An established strategy to confer structural uniqueness to two-stranded coiled coils is the use of buried polar Asn residues at position a, which imparts dimerization and conformational specificity at the expense of stability. Here we show that polar interactions involving buried position-a Lys residues that can interact favorably only with surface e' or g' Glu residues also impart structural uniqueness to a designed heterodimeric coiled coil with the nativelike properties of sigmoidal thermal and urea-induced unfolding transitions, slow hydrogen exchange and lack of ANS binding. The position-a Lys residues do not, however, confer a single preference for helix orientation, likely reflecting the ability of Lys at position a to from favorable interactions with g' or e' Glu residues in the parallel and antiparallel orientations, respectively. The Lys-Glu polar interaction is less destabilizing than the Asn-Asn a-->a' interaction, presumably reflecting a higher desolvation penalty associated with the completely buried polar position-a groups. Our results extend the range of approaches for two-stranded coiled-coil design and illustrate the role of complementing polar groups associated with buried and surface positions of proteins in protein folding and design.     

Identification by site-directed mutagenesis of two lysine residues in cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxin binding.

Utilizing site-directed mutagenesis and an Escherichia coli expression system for bovine cholesterol side chain cleavage cytochrome P450, lysine residues at 377 and 381 are found to play crucial roles in binding bovine adrenodoxin, required for transfer of electrons to mitochondrial P450s. These lysine residues are conserved among mitochondrial P450s and have been implicated previously by chemical modification studies as being important for adrenodoxin binding. In the present study, site-directed mutagenesis producing either neutral or positive amino acids at 377 or 381 has no effect on the structure of side chain cleavage cytochrome P450 as determined spectrally or on the enzymatic conversion of cholesterol to pregnenolone. However, the estimated Ks of adrenodoxin binding is increased approximately 150-600-fold depending on the particular mutation. Therefore these conserved positively charged residues in mitochondrial P450s are the key sites for adrenodoxin binding which is electrostatic in nature.     

Secretion of Escherichia coli chloramphenicol acetyltransferase by mammalian cells

We show here that expression of the Escherichia coli cat gene in mammalian cells results in accumulation of enzymatically active CAT in the culture media as well as in the cytoplasm. We call the extracellular product secreted CAT (sCAT). Three to four days after introduction of cat-expressing plasmids into mouse L cells by transient transfection, total extracellular sCAT activity exceeds total cytoplasmic CAT activity. As sCAT levels increase, substantially more CAT is found outside the cells than inside at later times. Comparison of different populations of cat-expressing cells shows that, at any given time, the level of sCAT is proportional to the level of intracellular CAT. Thus, assay of sCAT provides a convenient, non-invasive alternative to assay of intracellular CAT. The molecular sizes of sCAT and intracellular CAT are indistinguishable, suggesting that the protein is not cleaved or glycosylated during secretion. Several observations, including a lack of sensitivity to drugs which inhibit Golgi activity, suggest that CAT may be secreted via an unusual pathway.     

Identification of tissue transglutaminase-reactive lysine residues in glyceraldehyde-3-phosphate dehydrogenase

Polyglutamine domains are excellent substrates for tissue transglutaminase resulting in the formation of cross-links with polypeptides containing lysyl residues. This finding suggests that tissue transglutaminase may play a role in the pathology of neurodegenerative diseases associated with polyglutamine expansion. The glycolytic enzyme GAPDH previously was shown to tightly bind several proteins involved in such diseases. The present study confirms that GAPDH is an in vitro lysyl donor substrate of tissue transglutaminase. A dansylated glutamine-containing peptide was used as probe for labeling the amino-donor sites. SDS gel electrophoresis of a time-course reaction mixture revealed the presence of both fluorescent GAPDH monomers and high molecular weight polymers. Western blot analysis performed using antitransglutaminase antibodies reveals that tissue transglutaminase takes part in the formation of heteropolymers. The reactive amino-donor sites were identified using mass spectrometry. Here, we report that of the 26 lysines present in GAPDH, K191, K268, and K331 were the only amino-donor residues modified by tissue transglutaminase.     

Acetyl coenzyme A binding by chloramphenicol acetyltransferase. Hydrophobic determinants of recognition and catalysis.

The preponderance of nonpolar contacts between CoA and chloramphenicol acetyltransferase in the high resolution structure of the binary complex prompted a study of selected hydrophobic residues by site-directed mutagenesis and steady-state kinetic analysis. Substitutions of three aromatic residues were used to evaluate binding contacts with the adenine moiety of CoA (Tyr-178), the pantetheine arm of the coenzyme (Tyr-56), and the S-acyl substituent (Phe-33). For those substitutions at residues 56 and 178 that cannot promote alternative polar interactions there is a correlation between residue hydrophobicity and the free energy of formation of the binary and ternary complexes of acetyl-CoA and chloramphenicol acetyltransferase and of the transition-state complex. Substitutions at Tyr-178 destabilize all such complexes to approximately the same extent (uniform binding changes), whereas those at Tyr-56 and Phe-33 cause differential binding changes, having a greater effect on the transition state than on either of the other complexes with acetyl-CoA.