Structural and Biochemical Analysis Reveal Pirins to Possess Quercetinase Activity

Abstract

Pirin is a recently identified eukaryotic protein implicated in transcriptional activation and apoptosis. Homologues of Pirin are highly conserved in both prokaryotes and eukaryotes, but their function remains poorly understood. We present here the crystal structure of the yhhW gene product, a putative Pirin homologue, from Escherichia coli and confirm its structural similarity to Pirin. The YhhW protein displays a bicupin fold with a single N-terminal metal coordination site. Molecular surface comparisons of YhhW and Pirin with structurally similar proteins suggested quercetin as a potential ligand. We demonstrate that both bacterial and human Pirins have quercetinase activity, which is inhibited by the addition of typical inhibitors of the quercetin 2,3-dioxygenase reaction. We also demonstrate the release of carbon monoxide as a reaction product. This is the first report of enzymatic activity for any member of the Pirin family and may be an important connection to their roles in transcriptional regulation.Pirin is a recently identified protein that is localized to subnuclear dotlike structures in a wide variety of human tissues (1). Pirins are highly conserved among mammals, plants, fungi, and prokaryotes and have been classified as a subfamily of the cupin superfamily on the basis of both sequence and structural similarity (1, 2). The cupin superfamily is one of the most functionally diverse protein classes and includes both enzymatic and nonenzymatic members, ranging from isomerases and epimerases that are involved in the modification of cell wall carbohydrates to nonenzymatic storage proteins in plant seeds and transcriptional cofactors in humans (2). Cupin proteins typically maintain a highly conserved GX 5 HXHX 3,4 -EX 6 G metal-binding motif, along with a GX 5 PXGX 2 HX 3 N motif, and a conserved ␤-barrel fold. The structure of human Pirin (hPirin), 1 the only structure of a Pirin determined to date, also displays two cupin domains, consistent with other bicupin family members (3). The function of Pirin is not known, and no enzymatic activity has been described for any of the homologues. Early studies demonstrated the ability of hPirin to interact with the nuclear factor I/CCAAAT box transcription factor (NF-I) (1) and the oncoprotein B cell lymphoma 3-encoded (Bcl-3) in vivo (4), suggesting that hPirin may be involved in the modulation of NFI/CTF1 transcriptional activation or possibly in other nuclear processes, such as DNA replication or repair (1). As observed with hPirin and its homologues, proteins involved with such fundamental cellular processes are highly conserved throughout evolution. Recent studies of tumor metastasis in advanced colorectal cancer have also implicated Pirin in cellular responses to polysaccharide-K, a chemoimmunotherapeutic agent from mushrooms (5).Studies of hPirin homologues in plants have suggested a possible involvement of these proteins in programmed cell death, seed germination, and seedling development. The Lepirin gene from tomato exhibited dramatically increased mRN...

The TvPirin Gene Is Necessary for Haustorium Development in the Parasitic Plant Triphysaria versicolor

“…In Serratia marcescens, Pirin modifies pyruvate catabolism by interacting with pyruvate dehydrogenase (Soo et al, 2007). Pirins can also function catalytically as quercetin dioxygenases (Adams and Jia, 2005). The crystalline structure of human Pirin indicates two similar b-barrel domains arranged face to face with an iron cofactor within a cavity formed at the N-terminal domain, suggesting possible involvement in redox reactions (Pang et al, 2004).…”

Pirin Inhibits Cellular Senescence in Melanocytic Cells

“…A recent report shows that PIR controls melanoma cell migration through the transcriptional regulation of snail homolog 2, SNAI2 (previously SLUG). 16 Other reports described quercetinase enzymatic activity, 17 and regulation of apoptosis 18,19 and stress response, unveiling a high degree of cell-type and species specificity in PIR function.…”

An Insight into the Mechanism of Human Cysteine Dioxygenase

“…Besides CDO, the cupin superfamily includes several other dioxygenases from all three kingdoms, including pirin that was first characterized structurally by our group (21,22) and was shown to possess quercetin dioxygenase activity by Adams and colleagues (23). Distinct from the other dioxygenases, CDO catalyzes an unusual oxidation reaction in which two oxygen atoms are added to a single atom without C-C bond cleavage in the substrate.…”