Analysis of Hydrogen Bonding in Enzyme-Substrate Complexes of Chloramphenicol Acetyltransferase by Infrared Spectroscopy and Site-Directed Mutagenesis

Murray, Iain A.; Derrick, Jeremy P.; White, Andrew J. P.; Drabble, Kevin; Wharton, Christopher W.; Shaw, William V.

doi:10.1021/bi00199a003

Cited by 8 publications

(7 citation statements)

References 26 publications

(35 reference statements)

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“…From a methodological point of view, we demonstrate that competition experiments can be performed in the infrared spectral region, from which relative dissociation constants can be obtained without the need to develop a binding assay, and confirm that different binding modes of ligands can be detected as shown before (White and Wharton, 1990;Murray et al, 1994;Ryan and Baenziger, 1999;and reviewed in Wharton, 2000). Applications like this will soon gain importance in fundamental and applied research when mixing devices become commercially available that makes these experiments just as easy to perform in the infrared spectral region as in the visible spectral range.…”

Section: Discussionsupporting

confidence: 69%

TNP-AMP Binding to the Sarcoplasmic Reticulum Ca2+-ATPase Studied by Infrared Spectroscopy

Liu

Barth

2003

Biophysical Journal

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Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.

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Section: Discussionsupporting

confidence: 69%

TNP-AMP Binding to the Sarcoplasmic Reticulum Ca2+-ATPase Studied by Infrared Spectroscopy

Liu

Barth

2003

Biophysical Journal

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“…Compounds 6 , 14 , 15 and 16 , lacking the acetate moiety at C13 hydroxyl group, suffer a paulic acid migration to that position, thus becoming shunt products: 6-hydroxyl-13- O -paulyl-paulinone ( 6′ ), 13- O -deacetyl-13- O -paulyl-paulomycin E ( 14′ ), 13- O -deacetyl-13- O -paulyl-paulomycin B ( 15′ ) and 13- O -deacetyl-13- O -paulyl-paulomycin A ( 16′ ). Migration of acyl groups in aqueous solutions has been demonstrated to occur in other compounds such as betacyanins where glucose 6′- O -position is always favored [ 43 ], thuggacins that suffer acyl migrations of their lactone group [ 44 ], and chloramphenicol that undergoes an intra molecular rearrangement of an acetyl group from 3-hydroxyl to 1-hydroxyl group [ 45 ]. It has been demonstrated that acyl migration occurs to primary hydroxyl groups, which is the most stable position for acyl moieties [ 46 , 47 ].…”

Section: Discussionmentioning

confidence: 99%

New insights into paulomycin biosynthesis pathway in Streptomyces albus J1074 and generation of novel derivatives by combinatorial biosynthesis

et al. 2016

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BackgroundStreptomyces albus J1074 produces glycosylated antibiotics paulomycin A, B and E that derive from chorismate and contain an isothiocyanate residue in form of paulic acid. Paulomycins biosynthesis pathway involves two glycosyltransferases, three acyltransferases, enzymes required for paulic acid biosynthesis (in particular an aminotransferase and a sulfotransferase), and enzymes involved in the biosynthesis of two deoxysugar moieties: D-allose and L-paulomycose.ResultsInactivation of genes encoding enzymes involved in deoxysugar biosynthesis, paulic acid biosynthesis, deoxysugar transfer, and acyl moieties transfer has allowed the identification of several biosynthetic intermediates and shunt products, derived from paulomycin intermediates, and to propose a refined version of the paulomycin biosynthesis pathway. Furthermore, several novel bioactive derivatives of paulomycins carrying modifications in the L-paulomycose moiety have been generated by combinatorial biosynthesis using different plasmids that direct the biosynthesis of alternative deoxyhexoses.ConclusionsThe paulomycins biosynthesis pathway has been defined by inactivation of genes encoding glycosyltransferases, acyltransferases and enzymes involved in paulic acid and L-paulomycose biosynthesis. These experiments have allowed the assignment of each of these genes to specific paulomycin biosynthesis steps based on characterization of products accumulated by the corresponding mutant strains. In addition, novel derivatives of paulomycin A and B containing L-paulomycose modified moieties were generated by combinatorial biosynthesis. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. In addition, the pyruvate dehydrogenase system form by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. The activity of the novel paulomycin derivatives carrying modifications in the L-paulomycose moiety is lower than the original compounds pointing to some interesting structure–activity relationships.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0452-4) contains supplementary material, which is available to authorized users.

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“…Preparation of 3-Acetyl-Cm. 3-Acetyl-Cm was made and purified by the procedure described by Murray et al (1994).…”

Section: Methodsmentioning

confidence: 99%

“…Much is now known of the interaction of CAT and its substrates from (a) crystallographic studies of the binary complexes of enzyme and Cm or CoA at 1.75 and 2.4 A resolution, respectively (Leslie, 1990;Leslie et al, 1988) and (b) results of spectroscopic experiments (fluorescence, NMR, and IR) with CAT and its ligands in conjunction with structure-based mutagenesis (Ellis et al, 1991;Derrick et al, 1992;Murray et al, 1994). The choice of protein for the above studies, as well as for those described here, has been the type III enzyme (CATm) which is not only the sole naturally occurring CAT variant to yield diffraction quality crystals but is also the most competent catalytically.…”

mentioning

confidence: 99%

Kinetic mechanism of chloramphenicol acetyltransferase: the role of ternary complex interconversion in rate determination

Ellis¹,

Bagshaw²,

Shaw³

1995

Biochemistry

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Chloramphenicol acetyltransferase (CAT) catalyzes the acetyl-CoA-dependent acetylation of chloramphenicol (Cm) by a ternary complex mechanism and with a random order of addition of substrates. A closer examination of the mechanism of the reaction catalyzed by the type III CAT variant (CATIII) has included the measurement of the individual rate constants by stopped-flow fluorimetry at 5 degrees C. Under all conditions employed, product release from the binary complexes in both forward and reverse reactions was found to be too slow to account for the observed overall rate of turnover for the reaction. Additional, faster routes for product release are achieved via the formation of the nonproductive ternary complexes (CAT:3-acetyl-Cm:acetyl-CoA and CAT:CoA:Cm). The release of 3-acetyl-Cm from the binary complex is 5-fold slower than kcat (135 s-1 at 5 degrees C), whereas the dissociation rate constants of 3-acetyl-Cm from the ternary complexes with CoA and acetyl-CoA are 120 and 200 s-1, respectively. Arrhenius plots of dissociation rate constants indicate a slow release of products over a broad temperature range. Computer simulations based on the rate constants of CATIII applied to a ternary complex mechanism, assuming random order of substrate addition and product release, yielded nonlinear initial rates of product formation unless both nonproductive ternary complexes were included in the model. Simulated steady-state kinetic analyses based on the latter assumption yielded kinetic parameters that compared favorably with those determined experimentally.(ABSTRACT TRUNCATED AT 250 WORDS)

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Analysis of Hydrogen Bonding in Enzyme-Substrate Complexes of Chloramphenicol Acetyltransferase by Infrared Spectroscopy and Site-Directed Mutagenesis

Cited by 8 publications

References 26 publications

TNP-AMP Binding to the Sarcoplasmic Reticulum Ca2+-ATPase Studied by Infrared Spectroscopy

TNP-AMP Binding to the Sarcoplasmic Reticulum Ca2+-ATPase Studied by Infrared Spectroscopy

New insights into paulomycin biosynthesis pathway in Streptomyces albus J1074 and generation of novel derivatives by combinatorial biosynthesis

Kinetic mechanism of chloramphenicol acetyltransferase: the role of ternary complex interconversion in rate determination

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