title: Structure and catalytic mechanism of LigI: insight into the amidohydrolase enzymes of cog3618 and lignin degradation.
abstract: LigI from Sphingomonas paucimobilis catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate and 4-carboxy-2-hydroxymuconate in the degradation of lignin. This protein is a member of the amidohydrolase superfamily of enzymes. The protein was expressed in Escherichia coli and then purified to homogeneity. The purified recombinant enzyme does not contain bound metal ions, and the addition of metal chelators or divalent metal ions to the assay mixtures does not affect the rate of product formation. This is the first enzyme from the amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. The kinetic constants for the hydrolysis of PDC are 340 s(-1) and 9.8 × 10(6) M(-1) s(-1) (k(cat) and k(cat)/K(m), respectively). The pH dependence on the kinetic constants suggests that a single active site residue must be deprotonated for the hydrolysis of PDC. The site of nucleophilic attack was determined by conducting the hydrolysis of PDC in (18)O-labeled water and subsequent (13)C nuclear magnetic resonance analysis. The crystal structures of wild-type LigI and the D248A mutant in the presence of the reaction product were determined to a resolution of 1.9 Å. The C-8 and C-11 carboxylate groups of PDC are coordinated within the active site via ion pair interactions with Arg-130 and Arg-124, respectively. The hydrolytic water molecule is activated by the transfer of a proton to Asp-248. The carbonyl group of the lactone substrate is activated by electrostatic interactions with His-180, His-31, and His-33.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
authors.forename: Merlin Eric
authors.initials: ME
authors.lastname: Hobbs
authors.forename: Vladimir
authors.initials: V
authors.lastname: Malashkevich
authors.forename: Howard J
authors.initials: HJ
authors.lastname: Williams
authors.forename: Chengfu
authors.initials: C
authors.lastname: Xu
authors.forename: J Michael
authors.initials: JM
authors.lastname: Sauder
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Burley
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008031:Lignin / Q000378:metabolism*; D008958:Models, Molecular; D011487:Protein Conformation; D011753:Pyrones / Q000737:chemistry / Q000378:metabolism; D020578:Sphingomonas / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011753:Pyrones; C024153:alpha-pyrone-4,6-dicarboxylic acid; D008031:Lignin; D000581:Amidohydrolases