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The last step in cephalosporin C formation revealed: Crystal Structures of Deacetylcephalosporin C Acetyltransferase from Acremonium chrysogenum in Complexes with Reaction Intermediates
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. (Hajdu)
2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 377, no 3, 935-944 p.Article in journal (Refereed) Published
Description
Abstract [en]

Deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of cephalosporin C, a broad-spectrum beta-lactam antibiotic of large clinical importance. The acetyl transfer step has been suggested to be limiting for cephalosporin C biosynthesis, but has so far escaped detailed structural analysis. We present here the crystal structures of DAC-AT in complexes with reaction intermediates, providing crystallographic snapshots of the reaction mechanism. The enzyme is found to belong to the alpha/beta hydrolase class of acetyltransferases, and the structures support previous observations of a double displacement mechanism for the acetyl transfer reaction in other members of this class of enzymes. The structures of DAC-AT reported here provide evidence of a stable acyl - enzyme complex, thus underpinning a mechanism involving acetylation of a catalytic serine residue by acetyl coenzyme A, followed by transfer of the acetyl group to deacetylcephalosporin C through a suggested tetrahedral transition state.

Place, publisher, year, edition, pages
2008. Vol. 377, no 3, 935-944 p.
Keyword [en]
cephalosporin C, beta-lactam antibiotic biosynthesis, Acremonium chrysogenum, X-ray crystallography, acetyl transferase
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-96606DOI: 10.1016/j.jmb.2008.01.047ISI: 000255374200030PubMedID: 18279889OAI: oai:DiVA.org:uu-96606DiVA: diva2:171240
Available from: 2008-01-10 Created: 2008-01-10 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Evasion and Attack: Structural Studies of a Bacterial Albumin-binding Protein and of a Cephalosporin Biosynthetic Enzyme
Open this publication in new window or tab >>Evasion and Attack: Structural Studies of a Bacterial Albumin-binding Protein and of a Cephalosporin Biosynthetic Enzyme
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium Finegoldia magna. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule.

Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2008. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 382
Keyword
human serum albumin, GA module, albumin-binding, Finegoldia magna, deacetylcephalosporin C acetyltransferase, cephalosporin C, antibiotic, beta-lactam, biosynthesis, Acremonium chrysogenum, X-ray crystallography
National Category
Structural Biology
Identifiers
urn:nbn:se:uu:diva-8399 (URN)978-91-554-7062-3 (ISBN)
Public defence
2008-02-01, B22, Biomedical Centre, Husargatan 3, Uppsala, 13:00
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Available from: 2008-01-10 Created: 2008-01-10Bibliographically approved

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Valegård, Karin

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