Structure of Amidase from Pseudomonas aeruginosa Showing a Trapped Acyl Transfer Reaction Intermediate State*
- Jorge Andrade ‡ ,
- Amin Karmali § ,
- Maria A. Carrondo ‡ and
- Carlos Frazão ‡ 1
- ‡Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras, and the §Centro de Investigação de Engenharia Química e Biotecnologia, Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1, 1950-062 Lisboa, Portugal
- 1 To whom correspondence should be addressed. Tel.: 351-214469666; Fax: 351-214433644; E-mail: frazao{at}itqb.unl.pt.
Abstract
Microbial amidases belong to the thiol nitrilases family and have potential biotechnological applications in chemical and pharmaceutical industries as well as in bioremediation. The amidase from Pseudomonas aeruginosa isa6 × 38-kDa enzyme that catalyzes the hydrolysis of a small range of short aliphatic amides. The hereby reported high resolution crystallographic structure shows that each amidase monomer is formed by a globular four-layer αββα sandwich domain with an additional 81-residue long C-terminal segment. This wraps arm-in-arm with a homologous C-terminal chain of another monomer, producing a strongly packed dimer. In the crystal, the biological active homo-hexameric amidase is built grouping three such dimers around a crystallographic 3-fold axis. The structure also elucidates the structural basis for the enzyme activity, with the nitrilases catalytic triad at the bottom of a 13-Å deep, funnel-shaped pocket, accessible from the solvent through a narrow neck with 3-Å diameter. An acyl transfer intermediate, resulting from the purification protocol, was found bound to the amidase nucleophilic agent, Cys166. These results suggest that some pocket defining residues should undergo conformational shifts to allow substrates and products to access and leave the catalytic pocket, for turnover to occur.
Footnotes
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↵2 The abbreviation used is: SIRAS, single isomorphous replacement with anomalous scattering.
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↵3 G. M. Sheldrick, personal communication.
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The atomic coordinates and structure factors (code 2UXY and 2UXYSF) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
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↵* This work was supported by Research Grants 702 and 25/2003 from Fundação para a Ciência e Tecnologia and Instituto Politécnico de Lisboa, Portugal. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received February 2, 2007.
- Revision received March 19, 2007.
