Abstract
Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin-6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 Å resolution (PDB code: 2PAJ). This protein folds as a distorted (β/α)
8
-barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosyl homocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin-6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s
−1
, 8.0 μM, and 1.3 × 10
5
M
−1
s
−1
for
k
cat
,
K
m
, and
k
cat
/
K
m
, respectively. The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (PDB code: 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed based upon the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that hydrogen bond with the carbonyl oxygen at C4, a conserved threonine residue that hydrogen bonds with N5, and another conserved threonine residue that hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes which are predicted to deaminate isoxanthopterin.