Ligand recognition by chloroperoxidase using molecular interaction fields and quantum chemistry calculations

We recently reported that chloroperoxidase (CPO) from Caldariomyces fumago showed atypical kinetic behavior for the oxidation of 4,6 dimethyl dibenzothiophene (DMDBT). In this work, we undertake the theoretical study of DMDBT docking into CPO's active site, in order to clarify its binding capacity and affinity using molecular interaction fields and quantum mechanical procedure. The results revealed that CPO has two substrate binding sites with similar affinities for DMDBT. This finding suggests that the atypical kinetic behavior of CPO for the oxidation of DMDBT might be due to the simultaneous binding of two DMDBT molecules during its reaction cycle. Finally, we extend these results to carbazole, a nitrogen-containing PAH, through experimental and theoretical studies.     

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.     

Acetyl coenzyme A binding by chloramphenicol acetyltransferase: long-range electrostatic determinants of coenzyme A recognition.

The possible involvement of arginyl and lysyl side chains of chloramphenicol acetyltransferase (CAT) in binding coenzyme A (CoA) was studied by means of chemical modification, site-directed mutagenesis, variation in ionic strength, use of competitive inhibitors or substrate analogues, and X-ray crystallography. Unlike a number of enzymes, including citrate synthase, CAT does not employ specific ion pairs with the phosphoanionic centers of CoA to bind the acetyl donor, and arginyl residues play no role in recognition of the coenzyme. Although phenylglyoxal inactivates CAT reversibly, it does so by the formation of an unstable adduct with a thiol group, that of Cys-31 in the chloramphenicol binding site. The inhibitory effect of increasing ionic strength on kcat/Km(acetyl-CoA) can be explained by long-range electrostatic interactions between CoA and the epsilon-amino groups of Lys-54 and Lys-177, both of which are solvent-accessible. The epsilon-amino group of Lys-54 contributes 1.3 kcal.mol-1 to the binding of acetyl-CoA via interactions with both the 3'- and 5'-phosphoanions of CoA. Lys-177 contributes only 0.4 kcal.mol-1 to the productive binding of acetyl-CoA, mediated by long-range (approximately 14 A) interactions with the 5'-alpha- and -beta-phosphoanions of CoA. The combined energetic contribution of Lys-54 and Lys-177 to acetyl-CoA binding (1.7 kcal.mol-1) is less than that previously demonstrated (2.4 kcal.mol-1) for a simple hydrophobic interaction between Tyr-178 and the adenine ring of CoA (Day & Shaw, 1992).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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 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.     

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.     

Alternative binding modes for chloramphenicol and 1-substituted chloramphenicol analogues revealed by site-directed mutagenesis and X-ray crystallography of chloramphenicol acetyltransferase

Leucine-160 of chloramphenicol acetyltransferase (CAT) has been replaced by site-directed mutagenesis to investigate enzyme-ligand interactions at the 1-hydroxyl substituent of the substrate chloramphenicol. The consequences of the substitution of Leu-160 by glutamine and by phenylalanine were deduced from the steady-state kinetic parameters for acetyl transfer from acetyl-CoA to the 3-hydroxyl of chloramphenicol and its analogues 1-deoxychloramphenicol and 1-acetylchloramphenicol. The acetyl group of the latter, which is a substrate both in vivo and in vitro, could potentially bind in a similar position to the 1-hydroxyl of chloramphenicol, in close proximity to the side chain of Leu-160. In the case of Gln-160 CAT, large increases in Km for the three acetyl acceptors were accompanied by small decreases in kcat and in apparent affinity for acetyl-CoA. Such results are consistent with the introduction of the relatively hydrophilic amide in place of the delta-methyl groups of Leu-160. The kinetic properties of Phe-160 CAT were unexpected in that Km for each of the three acetyl acceptors was unchanged or reduced, compared to the equivalent parameters for the wild-type enzyme, whereas kcat fell significantly (44-83-fold) in each case. The ratios of specificity constants (kcat/Km) for the acetylation of chloramphenicol compared with the alternative acyl acceptors were similar for wild-type and mutant enzymes. As the residue substitutions for Leu-160 do not result in enhanced discrimination against the binding and acetylation of 1-acetylchloramphenicol, it appears unlikely that the 1-acetyl group binds to the CAT active site in the same position as that occupied by the 1-hydroxyl of chloramphenicol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the regulatory sequences needed for induction of the chloramphenicol acetyltransferase gene cat-86 by chloramphenicol and amicetin.

Induction of the chloramphenicol acetyltransferase gene cat-86 in Bacillus subtilis results from the activation of translation of cat-86 mRNA. The inducers, chloramphenicol and amicetin, are thought to enable ribosomes to destabilize a stem-loop structure in cat-86 mRNA that sequesters the ribosome binding site for the cat-86 coding sequence, designated RBS-3. The region of cat-86 mRNA which is 5' to the stem-loop contained two additional ribosome binding sites, RBS-1 and RBS-2, located 84 and 56 nucleotides, respectively, upstream from RBS-3. RBS-1 and RBS-2 were each followed by a potential translation initiation codon and a short open reading frame. Bal 31-generated deletions into the 5' end of the regulatory region that removed RBS-1 but did not enter RBS-2 caused a fourfold decrease in the uninduced and chloramphenicol-induced level of cat-86 expression and a more than 10-fold reduction in the amicetin-induced level of expression. Deletions that removed both RBS-1 and RBS-2 but did not enter the stem-loop abolished both chloramphenicol- and amicetin-inducible expression. These data indicate that RBS-2 and sequences 3' to RBS-2 are minimally essential to chloramphenicol induction. However, the presence of RBS-1 in the mRNA elevated the maximum level of expression obtained during chloramphenicol induction. These studies also demonstrate that induction of cat-86 by amicetin is highly dependent on RBS-1. To determine whether a correlation existed between RBS-1 and amicetin inducibility, we examined the sequence of the regulatory regions for two natural variants of cat-86, cat-66 and cat-57, which are chloramphenicol inducible but are very poorly induced by amicetin. Both contained nucleotide sequence differences from cat-86 in the vicinity of RBS-1 that would prevent translation of the leader peptide associated with RBS-1 in cat-86. In contrast, the regulatory regions got the three genes were virtually identical in the vicinity of RBS-2. These data indicate that efficient induction by amicetin requires sequences that are not essential for induction by chloramphenicol.     

Ligand recognition by purified human mannose receptor.

In this work we examine the carbohydrate binding properties of human placental mannose receptor (HMR) using a rapid and sensitive enzyme-linked immunosorbent microplate assay. The assay is based on the inhibition of binding of highly purified receptor to yeast mannan-coated 96-well plates. The specificity of ligand binding was inferred from the potency of different saccharides in blocking HMR binding to the mannan-coated wells. The relative inhibitory potency of monosaccharides was L-Fuc greater than D-Man greater than D-Glc greater than D-GlcNAc greater than Man-6-P much greater than D-Gal much greater than L-Rha much greater than GalNAc. The inhibitory potency of mannose increased by two orders of magnitude when linear oligomers were used. Oligomers containing alpha-1-3- and alpha-1-6-linked mannose residues were more inhibitory than those containing alpha-1-2- and alpha-1-4-linked mannoses. Linear or branched oligomannosides larger than three units did not have a significant influence on their inhibitory potency; rather, potency was found to decrease in comparison with oligomannosides with three units. Compared to linear oligomers, inhibition of binding was the best using branched mannose oligosaccharides, alpha-D-Man-bovine serum albumin conjugates, or mannan. A model is discussed in which branched ligand is bound to spatially distinct sites on the HMR.     

Ligand recognition by the vitamin D receptor.

Three-dimensional structure of the ligand binding domain (LBD) of the vitamin D receptor (VDR) docked with the natural ligand 1 alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] has been mostly solved by the X-ray crystallographic analysis of the deletion mutant (VDR-LBD Delta 165-215). The important focus, from now on, is how the VDR recognizes and interacts with potent synthetic ligands. We now report the docking models of the VDR with three functionally and structurally interesting ligands, 22-oxa-1,25-(OH)(2)D(3) (OCT), 20-epi-1,25-(OH)(2)D(3) and 20-epi-22-oxa-24,26,27-trihomo-1,25-(OH)(2)D(3). In parallel with the computational docking studies, we prepared twelve one-point mutants of amino acid residues lining the ligand binding pocket of the VDR and examined their transactivation potency induced by 1,25-(OH)(2)D(3) and these synthetic ligands. The results indicate that L233, R274, W286, H397 and Y401 are essential for holding the all ligands tested, S278 and Q400 are not important at all, and the importance of S237, V234, S275, C288 and H305 is variable depending on the side-chain structure of the ligands. Based on these studies, we suggested key structural factors to bestow the selective action on OCT and the augmented activities on 20-epi-ligands. Furthermore, the docking models coincided well with our proposed active space-region theory of vitamin D based on the conformational analyses of ligands.     

Purification and crystallization of Pseudomonas aeruginosa chloramphenicol acetyltransferase

A chloramphenicol acetyltransferase from Pseudomonas aeruginosa genomic DNA has been overexpressed, refolded, purified, and crystallized. Crystals suitable for a three-dimensional x-ray structure determination were obtained from solutions of polyethyleneglycol methyl ether 2000 containing NiCl₂ at pH 8.5. These crystals belong to the cubic space group P4_1/332 (a = 154.8 Å) and diffract x-rays to ≈3.2 Å resolution. Proteins 28:298–300, 1997. © 1997 Wiley-Liss Inc.     

Averaging interaction energies over homologs improves protein fold recognition in gapless threading.

Protein structure prediction is limited by the inaccuracy of the simplified energy functions necessary for efficient sorting over many conformations. It was recently suggested (Finkelstein, Phys Rev Lett 1998;80:4823-4825) that these errors can be reduced by energy averaging over a set of homologous sequences. This conclusion is confirmed in this study by testing protein structure recognition in gapless threading. The accuracy of recognition was estimated by the Z-score values obtained in gapless threading tests. For threading, we used 20 target proteins, each having from 20 to 70 homologs taken from the HSSP sequence base. The energy of the native structures was compared with the energy from 34 to 75 thousand of alternative structures generated by threading. The energy calculations were done with our recently developed Calpha atom-based phenomenological potentials. We show that averaging of protein energies over homologs reduces the Z-score from approximately -6.1 (average Z-score for individual chains) to approximately -8.1. This means that a correct fold can be found among 3 x 10(9) random folds in the first case and among 3 x 10(15) in the second. Such increase in selectivity is important for recognition of protein folds.