Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate

Lewendon, Ann; Murray, Iain A.; Kleanthous, Colin; Cullis, Paul M.; Shaw, William V.

doi:10.1021/bi00419a032

Cited by 62 publications

(77 citation statements)

References 33 publications

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“…By analogy with CAT, a prototype AT, H124 of PapA5 would be expected to act as the catalytic base (41). By the same criterion, D128 in PapA5 should be required as an active site stabilizer (38,42 (38,41). Mutational analysis of the proposed PapA5 catalytic site motif is consistent with the catalytic mechanism proposed for CAT and other ATs.…”

Section: Discussionsupporting

confidence: 52%

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Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

Onwueme

Ferreras

Buglino

et al. 2004

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

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Mycobacterium tuberculosis (Mt) produces complex virulence-enhancing lipids with scaffolds consisting of phthiocerol and phthiodiolone dimycocerosate esters (PDIMs). Sequence analysis suggested that PapA5, a so-called polyketide-associated protein (Pap) encoded in the PDIM synthesis gene cluster, as well as PapA5 homologs found in Mt and other species, are a subfamily of acyltransferases. Studies with recombinant protein confirmed that PapA5 is an acetyltransferase. Deletion analysis in Mt demonstrated that papA5 is required for PDIM synthesis. We propose that PapA5 catalyzes diesterification of phthiocerol and phthiodiolone with mycocerosate. These studies present the functional characterization of a Pap and permit inferences regarding roles of other Paps in the synthesis of complex lipids, including the antibiotic rifamycin

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Section: Discussionsupporting

confidence: 52%

“…1). Both motifs are present in ATs, where the fully conserved His and Asp residues are proposed to act as a catalytic base and an active site conformation stabilizer, respectively (36)(37)(38). We analyzed the activity of PapA5 variants PapH124A and PapD128A, with Ala substitutions in H124 (second His in the motif) and D128, respectively.…”

Section: Methodsmentioning

confidence: 99%

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

Onwueme

Ferreras

Buglino

et al. 2004

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

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“…The main chain is dislocated, and the network of stabilizing hydrogen bonds as well as the salt bridge between aspartate and arginine is interrupted. The replacement of Asp199 by alanine did not influence the activity dramatically but significantly increased the thermolability of the enzyme, which also underlines the proposed structure-stabilizing effect of the buried charge of Asp199 (177,183,184). The second substrate, acetyl-CoA, must approach the catalytic site through an alternative tunnel from a different direction than chloramphenicol, because the latter blocks the access to the active site in the pocket.…”

Section: Chloramphenicol Acetyltransferasementioning

confidence: 79%

Acyltransferases in Bacteria

Röttig

Steinbüchel

2013

Microbiol Mol Biol Rev

148

141

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SUMMARY Long-chain-length hydrophobic acyl residues play a vital role in a multitude of essential biological structures and processes. They build the inner hydrophobic layers of biological membranes, are converted to intracellular storage compounds, and are used to modify protein properties or function as membrane anchors, to name only a few functions. Acyl thioesters are transferred by acyltransferases or transacylases to a variety of different substrates or are polymerized to lipophilic storage compounds. Lipases represent another important enzyme class dealing with fatty acyl chains; however, they cannot be regarded as acyltransferases in the strict sense. This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms. On the basis of their studied or assumed mechanisms, most of the acyl-transferring enzymes can be divided into two groups. The majority of enzymes discussed in this review employ a conserved acyltransferase motif with an invariant histidine residue, followed by an acidic amino acid residue, and their catalytic mechanism is characterized by a noncovalent transition state. In contrast to that, lipases rely on completely different mechanism which employs a catalytic triad and functions via the formation of covalent intermediates. This is, for example, similar to the mechanism which has been suggested for polyester synthases. Consequently, although the presented enzyme types neither share homology nor have a common three-dimensional structure, and although they deal with greatly varying molecule structures, this variety is not reflected in their mechanisms, all of which rely on a catalytically active histidine residue.

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“…Replacement of His602 in E2p of E. coli by a cysteine residue abolished E2 activity without affecting other E2p functions [43]. On the other hand, the conversion of thc putative active-site His in E2 from Succhuronzyccs [45]. The mutation of the corresponding Asp residue in E2 from S. crrevisiae to Ala resulted in loss of the catalytic activity [44].…”

Section: A Poinl Mulution In the N-terminal Part Of The Catalytic Dommentioning

confidence: 99%

Site‐directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Schulze

Westphal

Boumans

et al. 1991

European Journal of Biochemistry

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(Receivcd May 13, 1991) -EJB 91 0615Site-directed mutagenesis was performed in the protease-sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (Elp) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex.Upon binding of peripheral components. the 24-subunit core of '4. vinelandii wild-type E2p dissociates into tetramers. Four E l p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E l p dimers.Large deletions of the protease-sensitive region of E2p resulted in a total loss of the E l p and E3 binding. A small deletion ( A P361-R362) or the point mutation K367Q in the protease-sensitive region did not influence E3 binding, but affected E l p binding strongly, although with excess Elp almost complete reconstitution was reached. For E2p with the point mutation R416D in the N-terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak Elp/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type 111 from Eschevichia coli.Deletion of the complete Ala + Pro-rich sequence between the protease-sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E l p or E3. A further deletion of 20 N-terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E l p binding was lost and, in contrast to the wild-type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E l p with the E l p sites and dissociation are linked processes.It is concluded that the binding site for E3 is located on the N-terminal part of the protease-sensitive region. In contrast, the binding site for E l p consists of two regions, one located on the protease-sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA [I]. It

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Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate

Cited by 62 publications

References 33 publications

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

Mycobacterial polyketide-associated proteins are acyltransferases: Proof of principle with Mycobacterium tuberculosis PapA5

Acyltransferases in Bacteria

Site‐directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii

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