High resolution structure and catalysis of O-acetylserine sulfhydrylase isozyme B from Escherichia coli

The crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 Å resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O-acet...

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Bibliographic Details
Published in:The FEBS journal 2007-10, Vol.274 (20), p.5382-5389
Main Authors: Zocher, Georg, Wiesand, Ulrich, Schulz, Georg E
Format: Article
Language:English
Subjects:
Binding Sites
Biochemistry
biosynthesis of l‐cysteine
Catalysis
Crystal structure
Crystallography, X-Ray
Cysteine Synthase - chemistry
Cysteine Synthase - metabolism
Diffraction
E coli
enzymatic assay
Enzymes
Escherichia coli - enzymology
homodimer asymmetry
Hydrogen-Ion Concentration
Isoenzymes - chemistry
Models, Molecular
Mutation
nonstandard l‐amino acids
Protein Conformation
Protein Subunits - chemistry
Pyridoxal Phosphate - chemistry
Spectrometry, Fluorescence
Spectrophotometry
X-rays
X‐ray diffraction
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Summary:The crystal structure of the dimeric O-acetylserine sulfhydrylase isozyme B from Escherichia coli (CysM), complexed with the substrate analog citrate, has been determined at 1.33 Å resolution by X-ray diffraction analysis. The C1-carboxylate of citrate was bound at the carboxylate position of O-acetylserine, whereas the C6-carboxylate adopted two conformations. The activity of the enzyme and of several active center mutants was determined using an assay based on O-acetylserine and thio-nitrobenzoate (TNB). The unnatural substrate TNB was modeled into the reported structure. The substrate model and the observed mutant activities may facilitate future protein engineering attempts designed to broaden the substrate spectrum of the enzyme. A comparison of the reported structure with previously published CysM structures revealed large conformational changes. One of the crystal forms contained two dimers, each of which comprised one subunit in a closed and one in an open conformation. Although the homodimer asymmetry was most probably caused by crystal packing, it indicates that the enzyme can adopt such a state in solution, which may be relevant for the catalytic reaction.
ISSN:1742-464X
1742-4658
DOI:10.1111/j.1742-4658.2007.06063.x