The crystal structure, mutagenesis, and activity studies reveal that patatin is a lipid acyl hydrolase with a Ser-Asp catalytic dyad.
Patatin is a nonspecific lipid acyl hydrolase that accounts for approximately 40% of the total soluble protein in mature potato tubers, and it has potent insecticidal activity against the corn rootworm. We determined the X-ray crystal structure of a His-tagged variant of an isozyme of patatin, Pat17, to 2.2 A resolution, employing SeMet multiwavelength anomalous dispersion (MAD) phasing methods. The patatin crystal structure has three molecules in the asymmetric unit, an R-factor of 22.0%, and an R(free) of 27.2% (for 10% of the data not included in the refinement) and includes 498 water molecules. The structure notably revealed that patatin has a Ser-Asp catalytic dyad and an active site like that of human cytosolic phospholipase A(2) (cPLA(2)) [Dessen, A., et al. (1999) Cell 97, 349-360]. In addition, patatin has a folding topology related to that of the catalytic domain of cPLA(2) and unlike the canonical alpha/beta-hydrolase fold. The structure confirms our site-directed mutagenesis and bioactivity data that initially suggested patatin possessed a Ser-Asp catalytic dyad. Alanine-scanning mutagenesis revealed that Ser77 and Asp215 were critical for both esterase and bioactivity, consistent with prior work implicating a Ser residue [Strickland, J. H., et al. (1995) Plant Physiol. 109, 667-674] and a Ser-Asp dyad [Hirschberg, H. J. H. B., et al. (2001) Eur. J. Biochem. 268, 5037-5044] in patatin's catalytic activity. The crystal structure aids the understanding of other structure-function relationships in patatin. Patatin does not display interfacial activation, a hallmark feature of lipases, and this is likely due to the fact that it lacks a flexible lid that can shield the active site.
Rydel
Timothy J
TJ
Monsanto Company, Chesterfield, Missouri 63017-1732, USA. timothy.j.rydel@monsanto.com
Williams
Jennifer M
JM
Krieger
Elysia
E
Moshiri
Farhad
F
Stallings
William C
WC
Brown
Sherri M
SM
Pershing
Jay C
JC
Purcell
John P
JP
Alibhai
Murtaza F
MF
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D001517:Coleoptera / Q000187:drug effects; D018360:Crystallography, X-Ray; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D007814:Larva; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000235:genetics; D010843:Pichia / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017433:Protein Structure, Secondary; D012694:Serine / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D010940:Plant Proteins; C043222:patatin protein, Solanum tuberosum; D001224:Aspartic Acid; D012694:Serine; D002265:Carboxylic Ester Hydrolases; D010741:Phospholipases A; D000409:Alanine