Crystal and molecular structure of chloramphenicol

Chatterjee, Chandranath; Dattagupta, J.K.; Saha, N. N.; Muller, Kina

doi:10.1007/bf01200521

Cited by 10 publications

(28 citation statements)

References 9 publications

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“…Only five of these residues are strictly conserved among all known CAT sequences, but the observed substitutions are always conservative. The conformation of chloramphenicol is very similar to that seen in the single-crystal structure (27) (28). The major difference is that the primary (C-3) hydroxyl of enzyme-bound chloramphenicol (the acetyl acceptor in the forward reaction) adopts an alternative staggered conformation to that seen in the crystal structure of the antibiotic.…”

Section: Fph0bmentioning

confidence: 53%

Structure of chloramphenicol acetyltransferase at 1.75-A resolution.

Leslie

Moody

Shaw

1988

Proc. Natl. Acad. Sci. U.S.A.

166

137

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Chloramphenicol acetyltransferase [acetyl-CoA:chloramphenicol 03-acetyltransferase; EC 2.3.1.28] is the enzyme responsible for high-level bacterial resistance to the antibiotic chloramphenicol. It catalyzes the transfer of an acetyl group from acetyl CoA to the primary hydroxyl of chloramphenicol. The x-ray crystallographic structure of the type m variant enzyme from Escherichia cofl has been determined and refined at 1.75-resolution. The enzyme is a trimer of identical subunits with a distinctive protein fold. Structure of the trimer is stabilized by a 13-pleated sheet that extends from one subunit to the next. The active site is located at the subunit interface, and the binding sites for both chloramphenicol and CoA have been characterized. Substrate binding is unusual in that the two substrates approach the active site via clefts on opposite molecular "sides." A histidine residue previously implicated in catalysis is appropriately positioned to act as a general base catalyst in the reaction.Resistance to antibiotics in pathogenic bacteria is an increasingly common phenomenon, which has serious implications for clinical medicine. The resistance is frequently achieved by enzymatically catalyzed covalent modification of the drug. For chloramphenicol, inactivation is achieved by 0-acetylation. Because the modified drug no longer binds to a bacterial ribosome, which is its normal site of action, the drug loses its effect as an antibiotic (1). The enzyme responsible for this acetylation is chloramphenicol acetyltransferase (CAT) (acetyl-CoA:chloramphenicol acetyltransferase; EC 2.3.1.28), which catalyzes transfer of an acetyl group from acetyl CoA to the primary hydroxyl (C-3) of chloramphenicol (Cm) (2-4).The CAT gene is commonly, but not exclusively, plasmidborne in natural isolates and has been found to be a component of plasmids conferring multiple drug resistance, especially in Gram-negative bacteria and the Enterobacteriaceae, in particular (5). Amino acid sequences of several variants of CAT from both Gram-positive and Gram-negative bacteria have been determined (6-11). All known variants have similar subunit molecular weights (Mr 25,000) and are highly homologous, indicating similar tertiary structure. This conclusion had been inferred from earlier studies of hybrids formed in vivo and in vitro between naturally occurring variants (12, 13 (15), and the type III variant, which has been studied by kinetic and chemical methods (16,17) and which is currently the only variant to yield crystals suitable for x-ray diffraction studies (18).We report here the three-dimensional structures of two binary complexes of CAT, one with the substrate chloramphenicol bound and the second with bound CoA (a product of the forward reaction and substrate for the reverse reaction). Structure Determination Crystals of the binary complex of the enzyme with bound chloramphenicol were obtained by microdialysis ofprotein (5 mg/ml) in 10 mM Mes, pH 6.3, against 2% (vol/vol) 2-methyl-2,4-pentandiol/10 mM Mes, pH 6.3/1 mM chloram...

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Section: Fph0bmentioning

confidence: 53%

Structure of chloramphenicol acetyltransferase at 1.75-A resolution.

Leslie

Moody

Shaw

1988

Proc. Natl. Acad. Sci. U.S.A.

166

137

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“…Colorless, needle-shaped crystals of CLM were obtained by slow evaporation of an aqueous solution of the compound at room temperature. It crystallizes in the orthorhombic 17 Fig. 1.…”

Section: Chloramphenicol Samplementioning

confidence: 99%

NIR‐FT Raman, FT‐IR and surface‐enhanced Raman scattering spectra, with theoretical simulations on chloramphenicol

Sajan¹,

Sockalingum

Manfait

et al. 2008

J Raman Spectroscopy

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Chloramphenicol (CLM), originally derived from the bacterium Streptomyces venezuelae, is an inhibitor of bacterial ribosomal peptidyl transferase activity. The near infrared Fourier transform (NIR-FT) Raman, surface-enhanced Raman spectroscopy (SERS) and Fourier transform infrared (FT-IR) spectral analyses of CLM, a potential antibacterial drug for the treatment of typhoid fever, were carried out along with density functional computations. The vibrational spectral analysis reveals that the CH 2 asymmetric and symmetric stretching modes are shifted to higher wavenumbers than the computed values, owing to the electronic effects resulting from induction of methylene group with the adjacent electronegative atom. The lowering of C O stretching wavenumber is due to the presence of the strong electronegative atom, nitrogen, attached to the carbonyl carbon, causing large degree of molecular p-electron delocalization and redistribution of electrons, which weakens the C O bond. The absence of a C-H stretching vibration and the observed C-H out-of-plane bending modes suggest that the CLM molecule may be adsorbed in a flat orientation with respect to the silver surface.

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“…The conformational analysis of CHL should be of high interest to broad synthetic/medicinal chemists and chemical biologists who aim for design and development of new antibiotics [4,5]. However, since the conformational behavior of pure CHL was investigated several decades ago using only experimental techniques and quite outdated computational methods [6,7,8,9,10,11,12], currently there is no consensus on the actual conformation of free CHL molecules in solution [2]. Jardetzky [9] concluded by means of NMR spectroscopy the presence of a unique intramolecularly hydrogen bonded CHL conformer in solvents of different polarity ranging from acetone to water.…”

Section: Introductionmentioning

confidence: 99%

“…Hahn claimed a definitive study in view of these contradictory reports [2]. The lack of consensus also extends to the solid phase: Chatterjee [7] proposed an intramolecularly hydrogen bonded conformer in which the primary hydroxyl group acts as hydrogen bond donor, but Acharya [8] reported the secondary hydroxyl as the actual hydrogen bond donor. None of these crystalline structures has been discarded, and both are considered in the current literature [2].…”

Section: Introductionmentioning

confidence: 99%

“…This paper revisits the conformational behavior of CHL in the crystalline phase and in the free molecules in solution, solving the contradictory results found in the literature and stablishing the correct conformational features of CHL. FTIR spectroscopy is used to discriminate between the two crystallines structures currently accepted [7,8]. The conformational behavior of free CHL molecules in solution is analyzed using the Onsager self-consistent reaction field (SCRF) model.…”

Section: Introductionmentioning

confidence: 99%

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