Structure, activity, and substrate selectivity of the Orf6 thioesterase from Photobacterium profundum.
Enviado por Abel Baerga-Ortiz el
Título | Structure, activity, and substrate selectivity of the Orf6 thioesterase from Photobacterium profundum. |
Publication Type | Journal Article |
Year of Publication | 2013 |
Autores | Rodríguez-Guilbe, M, Oyola-Robles, D, Schreiter, ER, Baerga-Ortiz, A |
Journal | J Biol Chem |
Volume | 288 |
Issue | 15 |
Pagination | 10841-8 |
Date Published | 2013 Apr 12 |
ISSN | 1083-351X |
Palabras clave | Bacterial Proteins, Crystallography, X-Ray, Open Reading Frames, Palmitoyl Coenzyme A, Photobacterium, Protein Multimerization, Protein Structure, Quaternary, Substrate Specificity, Thiolester Hydrolases |
Abstract | Thioesterase activity is typically required for the release of products from polyketide synthase enzymes, but no such enzyme has been characterized in deep-sea bacteria associated with the production of polyunsaturated fatty acids. In this work, we have expressed and purified the Orf6 thioesterase from Photobacterium profundum. Enzyme assays revealed that Orf6 has a higher specific activity toward long-chain fatty acyl-CoA substrates (palmitoyl-CoA and eicosapentaenoyl-CoA) than toward short-chain or aromatic acyl-CoA substrates. We determined a high resolution (1.05 Å) structure of Orf6 that reveals a hotdog hydrolase fold arranged as a dimer of dimers. The putative active site of this structure is occupied by additional electron density not accounted for by the protein sequence, consistent with the presence of an elongated compound. A second crystal structure (1.40 Å) was obtained from a crystal that was grown in the presence of Mg(2+), which reveals the presence of a binding site for divalent cations at a crystal contact. The Mg(2+)-bound structure shows localized conformational changes (root mean square deviation of 1.63 Å), and its active site is unoccupied, suggesting a mechanism to open the active site for substrate entry or product release. These findings reveal a new thioesterase enzyme with a preference for long-chain CoA substrates in a deep-sea bacterium whose potential range of applications includes bioremediation and the production of biofuels. |
DOI | 10.1074/jbc.M112.446765 |
Alternate Journal | J. Biol. Chem. |
PubMed ID | 23430744 |
PubMed Central ID | PMC3624464 |
Grant List | 2G12-RR003051 / RR / NCRR NIH HHS / United States 8G12-MD007600 / MD / NIMHD NIH HHS / United States G12 MD007600 / MD / NIMHD NIH HHS / United States G12 RR003051 / RR / NCRR NIH HHS / United States R25 GM061838 / GM / NIGMS NIH HHS / United States R25GM061838 / GM / NIGMS NIH HHS / United States / / Howard Hughes Medical Institute / United States |