High‐resolution structure of the M14‐type cytosolic carboxypeptidase from Burkholderia cenocepacia refined exploiting PDB_REDO strategies. (1st February 2014)

• Record Type: Journal Article
• Authors: Rimsa, Vadim; Eadsforth, Thomas C.; Joosten, Robbie P.; Hunter, William N.
• Is Part Of: Acta crystallographica. Volume 70:Part 2(2014:Feb.)
• Page Start: 279
• Page End: 289
• Publication Date: 2014-02-01
• Subjects: Biomolecules -- Structure -- Periodicals; Physical biochemistry -- Periodicals; X-ray crystallography -- Periodicals; Crystallography -- Periodicals; 572
• DOI: 10.1107/S1399004713026801
• Languages: English
• ISSNs: 0907-4449

Abstract

<abstract abstract-type="main" xml:lang="en"> <title> <x xml:space="preserve">Abstract</x> </title> <p>A potential cytosolic metallocarboxypeptidase from <italic>Burkholderia cenocepacia</italic> has been crystallized and a synchrotron‐radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 Å resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high‐throughput automated protocols embedded in <italic>PDB_REDO</italic> were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N‐terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C‐terminal domain, which carries the Zn<sup>2+</sup>‐containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The <italic>B. cenocepacia</italic> protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide‐bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn<sup>2+</sup>, where an acetate ion is identified, suggests recognition of the carboxy‐terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1′ recognition subsite that suggests specificity towards an acidic substrate.</p> </abstract>