Abstract
LigI from
Sphingomonas paucimobilis
catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate (OMA) and 4-carboxy-2-hydroxymuconate (CHM) in the degradation of lignin. This protein is a member of the amidohydrolase superfamily of enzymes. The protein was expressed in
E. 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
for the values of
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 NMR 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 a proton transfer to Asp-248. The carbonyl group of the lactone substrate is activated by electrostatic interactions with His-180, His-31 and His-33.