Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity.

The biological role of the bacterial chloramphenicol (Chl)-resistance enzyme, chloramphenicol acetyltransferase (CAT), has seen renewed interest due to the resurgent use of Chl against multi-drug-resistant microbes. This looming threat calls for more rationally designed antibiotic derivatives that have improved antimicrobial properties and reduced toxicity in humans. Herein, we utilize native ion mobility spectrometry-mass spectrometry (IMS-MS) to investigate the gas-phase structure and thermodynamic stability of the type I variant of CAT from Escherichia coli (EcCATI) and several EcCATI:ligand-bound complexes. EcCATI readily binds multiple Chl without incurring significant changes to its gas-phase structure or stability. A non-hydrolyzable acetyl-CoA derivative (S-ethyl-CoA, S-Et-CoA) was used to kinetically trap EcCATI and Chl in a ternary, ligand-bound state (EcCATI:S-Et-CoA:Chl). Using collision-induced unfolding (CIU)-IMS-MS, we find that Chl dissociates from EcCATI:S-Et-CoA:Chl complexes at low collision energies, while S-Et-CoA remains bound to EcCATI even as protein unfolding occurs. Gas-phase binding constants further suggest that EcCATI binds S-Et-CoA more tightly than Chl. Both ligands exhibit negative cooperativity of subsequent ligand binding in their respective binary complexes. While we observe no significant change in structure or stability to EcCATI when bound to either or both ligands, we have elucidated novel gas-phase unfolding and dissociation behavior and provided a foundation for further characterization of alternative substrates and/or inhibitors of EcCATI.

Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness.

In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species.

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid-mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.

Heme interacts with histidine- and tyrosine-based protein motifs and inhibits enzymatic activity of chloramphenicol acetyltransferase from Escherichia coli.

BACKGROUND: The occurrence of free organismal heme can either contribute to serious diseases or beneficially regulate important physiological processes. Research on transient binding to heme-regulatory motifs (HRMs) in proteins resulted in the discovery of numerous Cys-based, especially Cys-Pro (CP)-based motifs. However, the number of His- and Tyr-based protein representatives is comparatively low so far, which is in part caused by a lack of information regarding recognition and binding requirements. METHODS: To understand transient heme association with such motifs on the molecular level, we analyzed a set of 44 His- and Tyr-based peptides using UV-vis, resonance Raman, cw-EPR and 2D NMR spectroscopy. RESULTS: We observed similarities with Cys-based sequences with respect to their spectral behavior and complex geometries. However, significant differences regarding heme-binding affinities and sequence requirements were also found. Compared to Cys-based peptides and proteins all sequences investigated structurally display increased flexibility already in the free-state, which is also maintained upon heme association. The acquired knowledge allowed for identification and prediction of a His-based HRM in chloramphenicol acetyltransferase from Escherichia coli as potential heme-regulated protein. The enzyme's heme-interacting capability was studied, and revealed an inhibitory effect of heme on the protein activity with an IC50 value of 57.69±4.37 µM. CONCLUSIONS: It was found that heme inhibits a bacterial protein carrying a potential His-based HRM. This finding brings microbial proteins more into focus of regulation by free heme. GENERAL SIGNIFICANCE: Understanding transient binding and regulatory action of heme with bacterial proteins, being crucial for survival, might promote new strategies for the treatment of bacterial infections.

Regioselective Acetylation of C21 Hydroxysteroids by the Bacterial Chloramphenicol Acetyltransferase I.

Chloramphenicol acetyltransferase I (CATI) detoxifies the antibiotic chloramphenicol and confers a corresponding resistance to bacteria. In this study we identified this enzyme as a steroid acetyltransferase and designed a new and efficient Escherichia-coli-based biocatalyst for the regioselective acetylation of C21 hydroxy groups in steroids of pharmaceutical interest. The cells carried a recombinant catI gene controlled by a constitutive promoter. The capacity of the whole-cell system to modify different hydroxysteroids was investigated, and NMR spectroscopy revealed that all substrates were selectively transformed into the corresponding 21-acetoxy derivatives. The biotransformation was optimized, and the reaction mechanism is discussed on the basis of a computationally modeled substrate docking into the crystal structure of CATI.

Thermoadaptation-directed evolution of chloramphenicol acetyltransferase in an error-prone thermophile using improved procedures.

Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.

Versatile synthesis of probes for high-throughput enzyme activity screening.

Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination.

The structural basis for substrate versatility of chloramphenicol acetyltransferase CATI.

Novel antibiotics are needed to overcome the challenge of continually evolving bacterial resistance. This has led to a renewed interest in mechanistic studies of once popular antibiotics like chloramphenicol (CAM). Chloramphenicol acetyltransferases (CATs) are enzymes that covalently modify CAM, rendering it inactive against its target, the ribosome, and thereby causing resistance to CAM. Of the three major types of CAT (CAT(I-III)), the CAM-specific CAT(III) has been studied extensively. Much less is known about another clinically important type, CAT(I). In addition to inactivating CAM and unlike CAT(III), CAT(I) confers resistance to a structurally distinct antibiotic, fusidic acid. The origin of the broader substrate specificity of CAT(I) has not been fully elucidated. To understand the substrate binding features of CAT(I), its crystal structures in the unbound (apo) and CAM-bound forms were determined. The analysis of these and previously determined CAT(I)-FA and CAT(III)-CAM structures revealed interactions responsible for CAT(I) binding to its substrates and clarified the broader substrate preference of CAT(I) compared to that of CAT(III).

Facile synthesis of metal-chelating magnetic nanoparticles by exploiting organophosphorus coupling.

A new method is described for facile synthesis of metal-chelating magnetic nanoparticles by simply mixing iron oxide nanoparticles with a bifunctional organophosphorus compound, N-(phosphonomethyl)iminodiacetic acid (PM-IDA), in aqueous solution. On charging with nickel ions, the PM-IDA functionalized iron oxide nanoparticles exhibited high His-tag protein binding capacity (0.21 and 0.58 mg/mg for His-tagged green fluorescent protein and chloramphenicol acetyltransferase, respectively) and were successfully used to purify these proteins from bacterial cell extracts to high purity in a single step. Although other synthetic schemes for metal-chelating magnetic nanoparticles have been reported, the method described here is markedly simpler and involves only low-cost reagents.

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.

Protein design through systematic catalytic loop exchange in the (beta/alpha)8 fold.

Protein engineering by directed evolution has proven effective in achieving various functional modifications, but the well-established protocols for the introduction of variability, typically limited to random point mutations, seriously restrict the scope of the approach. In an attempt to overcome this limitation, we sought to explore variant libraries with richer diversity at regions recognized as functionally important through an exchange of natural components, thus combining design with combinatorial diversity. With this approach, we expected to maintain interactions important for protein stability while directing the introduction of variability to areas important for catalysis. Our strategy consisted in loop exchange over a (beta/alpha)(8) fold. Phosphoribosylanthranilate isomerase was chosen as scaffold, and we investigated its tolerance to loop exchange by fusing variant libraries to the chloramphenicol acetyl transferase coding gene as an in vivo folding reporter. We replaced loops 2, 4, and 6 of phosphoribosylanthranilate isomerase with loops of varied types and sizes from enzymes sharing the same fold. To allow for a better structural fit, saturation mutagenesis was adopted at two amino acid positions preceding the exchanged loop. Our results showed that 30% to 90% of the generated mutants in the different libraries were folded. Some variants were selected for further characterization after removal of chloramphenicol acetyl transferase gene, and their stability was studied by circular dichroism and fluorescence spectroscopy. The sequences of 545 clones show that the introduction of variability at "hinges" connecting the loops with the scaffold exhibited a noticeable effect on the appearance of folded proteins. Also, we observed that each position accepted foreign loops of different sizes and sequences. We believe our work provides the basis of a general method of exchanging variably sized loops within the (beta/alpha)(8) fold, affording a novel starting point for the screening of novel activities as well as modest diversions from an original activity.

Development of an in vitro cell culture assay system for measuring the activation of a neighbouring gene by the retroviral vector.

BACKGROUND: The use of retroviral vectors has shown an actual clinical benefit in a few inherited diseases. However, the occurrence of cases of leukemia after the X-SCID gene therapy trial raised concerns about the safety of insertional mutagenesis inherent to the biology of the retrovirus. Although the retrovirus has long been known to integrate into the host chromosome, and thus have the potential to activate the nearby gene, there has been no convenient method of studying or assaying such a cis-activation phenomenon. METHODS: In the present study, we report an in vitro assay system in which the effect of retroviral integration on the expression of the neighbouring gene can be studied. In this system, a retroviral vector and the neighbouring reporter gene were constructed in a single plasmid as if it had integrated into the chromosome. RESULTS: Using this assay, we found that the full-length long terminal repeat (LTR) could indeed activate the neighbouring gene expression from a distance and the magnitude of its activation was highly increased when this LTR was placed in the vicinity of the transcription start site of the gene, whereas the truncated LTR exerted little influence. CONCLUSIONS: This assay system might provide a useful tool for selecting the appropriate vector structure, as well as for studying the molecular mechanism underlying the cis-activation by the viral LTR.

Monitoring of Leuconostoc mesenteroides DRC starter in fermented vegetable by random integration of chloramphenicol acetyltransferase gene.

In 2004, Leuconostoc mesenteroides DRC was first used as a starter culture for achieving higher organoleptic effects in Korean kimchi manufacture. For a better understanding of starter growth in a mixed culture system, and for predicting starter predominance in kimchi, a monitoring system for the starter was established. The chloramphenicol resistance marker gene (cat) was randomly integrated into chromosomal DNA of L. mesenteroides DRC using a viral transposon and transposase. The DRC mutant, tDRC2, had a similar growth pattern to the host strain, with no major alteration in phenotypic characteristics. The mutant strain was inoculated into real kimchi, and monitoring of the starter population was successfully achieved. The overall predominance of Leuconostoc in kimchi inoculated with DRC followed the general growth pattern of this genus during kimchi fermentation. Our results also demonstrate the competitive ability of the DRC starter against Leuconostoc from natural flora, maintaining its predominance above 88% during the whole fermentation period. Based on this experiment, the random gene integration method using a transposon was shown to be of utility in transferring any commercial starter into a selectable and monitorable strain for simulation purposes.

Analysis of the distribution of functionally relevant rare codons.

BACKGROUND: The substitution of rare codons with more frequent codons is a commonly applied method in heterologous gene expression to increase protein yields. However, in some cases these substitutions lead to a decrease of protein solubility or activity. To predict these functionally relevant rare codons, a method was developed which is based on an analysis of multisequence alignments of homologous protein families. RESULTS: The method successfully predicts functionally relevant codons in fatty acid binding protein and chloramphenicol acetyltransferase which had been experimentally determined. However, the analysis of 16 homologous protein families belonging to the alpha/beta hydrolase fold showed that functionally rare codons share no common location in respect to the tertiary and secondary structure. CONCLUSION: A systematic analysis of multisequence alignments of homologous protein families can be used to predict rare codons with a potential impact on protein expression. Our analysis showed that most genes contain at least one putative rare codon rich region. Rare codons located near to those regions should be excluded in an approach of improving protein expression by an exchange of rare codons by more frequent codons.

Fluorinated chloramphenicol acetyltransferase thermostability and activity profile: improved thermostability by a single-isoleucine mutant.

A lysate-based thermostability and activity profile is described for chloramphenicol acetyltransferase (CAT) expressed in trifluoroleucine, T (CAT T). CAT and 13 single-isoleucine CAT mutants were expressed in medium supplemented with T and assayed for thermostability on cell lysates. Although fluorinated mutants, L82I T and L208I T, showed losses in thermostability, the L158I T fluorinated mutant demonstrated an enhanced thermostability relative to CAT T. Further characterization of L158I T suggested that T at position 158 contributed to a portion of the observed loss in thermostability upon global fluorination.

Structure and dynamics of pin1 during catalysis by NMR.

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the microsecond-to-millisecond timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidylprolyl isomerase (Pin1) catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer's disease. (15)N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of a substrate, suggesting that this area is already "primed" for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intramolecular nuclear Overhauser enhancements. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed the distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.

Cell-free synthesis of zinc-binding proteins.

Cell-free protein synthesis has become one of the standard methods for protein expression. The cell-free method is suitable for the synthesis of a protein that requires a ligand for its enzymatic activity and/or structure formation and stabilization, since it is an open system, which allows us to add the proper ligand to the reaction mixture. A large number of proteins that require zinc for their function are involved in diverse cellular processes, including transcription, DNA replication, metabolism, and cell signaling. In this study, we analyzed the effects of zinc on the cell-free synthesis of plant-specific zinc-binding transcription factors. The solubility and/or stability of the proteins were significantly increased in the presence of the proper concentration of zinc during the cell-free reaction. NMR analyses confirmed that correctly folded proteins were synthesized by the cell-free method. These results indicate that the cell-free method can be used to synthesize correctly folded and functional zinc-binding proteins.

Influence of global fluorination on chloramphenicol acetyltransferase activity and stability.

Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.

Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase.

A method for general protein biotinylation by enzymatic means has been developed. A mutant form (R118G) of the biotin protein ligase (BirA) of Escherichia coli is used and biotinylation is thought to proceed by chemical acylation of protein lysine side chains by biotinoyl-5'-AMP released from the mutant protein. Bovine serum albumin, chloramphenicol acetyltransferase, immunoglobulin chains and RNAse A as well as a large number of E. coli proteins have been biotinylated. The biotinylation reaction is proximity dependent in that the extent of biotinylation is much greater when the ligase is coupled to the acceptor protein than when the acceptor is free in solution. This is presumably due to rapid hydrolysis of the acylation agent, biotinoyl-5'-AMP. Therefore, when the mutant ligase is attached to one partner involved in a protein-protein interaction, it can be used to specifically tag the other partner with biotin, thereby permitting facile detection and recovery of the proteins by existing avidin/streptavidin technology.