Richard Hall - Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase

Version 1

      Publication Details (including relevant citation   information):

      Hall, Richard S.; Agarwal, Rakhi; Hitchcock, Daniel; Sauder, J.   Michael; Burley, Stephen K.; Swaminathan, Subramanyam; and   Raushel, Frank M..  Discovery and Structure Determination of   the Orphan Enzyme Isoxanthopterin Deaminase.  Biochemistry   (2010), 49(20), 4374-4382.  ISSN: 0006-2960.  DOI:   10.1021/bi100252s.

      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 (Protein   Data Bank entry   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-adenosylhomocysteine, 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 × 105 M−1 s−1  (kcat, Km, and   kcat/Km, respectively).   The active site of Sgx9339a most closely resembles the active   site for 8-oxoguanine deaminase (Protein Data Bank entry   2UZ9). A model for substrate recognition of isoxanthopterin   by Sgx9339a was proposed on the basis of 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 form hydrogen bonds with the carbonyl   oxygen at C4, a conserved threonine residue that forms hydrogen   bonds with N5, and another conserved threonine residue that forms   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.

      Address (URL): http://pubs.acs.org/doi/full/10.1021/bi100252s