Engineering cysteine mutants to obtain crystallographic phases with a cutinase from Fusarium solani pisi.
Cutinases are extracellular enzymes involved in the disruption of cutine, an insoluble polyester which covers the surface of plants. They belong to a class of serine esterases that are able to hydrolyse fatty acid esters and emulsified triglycerides as efficiently as lipases, but without displaying interfacial activation. Classical crystallographic methods for obtaining heavy-atom derivatives failed, so the cutinase structure has been solved exclusively by the multiple isomorphous replacement method using four Hg derivatives obtained from mutants S4C, S92C, S120C and S129C. Two of these derivatives behaved as expected: (i) the cys mutant of the catalytic Ser S120C, located at the surface of the active site pocket, leads to a good derivative; and (ii) the Hg atom of the derivative obtained with the S92C mutant is completely accessible to the solvent and occupies two alternative positions--consequently a poor derivative results. In contrast, two mutants show an unexpected behaviour: (i) the Hg atom in the S129C mutant was completely buried 10 A below the protein surface and yielded the best derivative; and (ii) a poor quality derivative was obtained with the S4C mutant. Cys 4 belongs to the disordered propeptide 1-16. The Cys 4 bound Hg atom is located in front of the Asp58 side chain, but neither Cys4 nor parts of the propeptide are clearly visible in the electron density maps of the derivative structure.
Martinez
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Laboratoire de Cristallographie et Cristallisation des Macromolécules biologiques, URA232, CNRS Faculté de Médecine Nord, Marseille, France.
de Geus
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Stanssens
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Lauwereys
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Cambillau
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D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics*; D003460:Crystallization; D003545:Cysteine; D004247:DNA / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009941:Organomercury Compounds / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D014961:X-Ray Diffraction
D005656:Fungal Proteins; D009941:Organomercury Compounds; D011993:Recombinant Fusion Proteins; D004247:DNA; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D003545:Cysteine