The first aspartic acid of the DQxD motif for human UDP-glucuronosyltransferase 1A10 interacts with UDP-glucuronic acid during catalysis.
All UDP-glucuronosyltransferase enzymes (UGTs) share a common cofactor, UDP-glucuronic acid (UDP-GlcUA). The binding site for UDP-GlcUA is localized to the C-terminal domain of UGTs on the basis of amino acid sequence homology analysis and crystal structures of glycosyltransferases, including the C-terminal domain of human UGT2B7. We hypothesized that the (393)DQMDNAK(399) region of human UGT1A10 interacts with the glucuronic acid moiety of UDP-GlcUA. Using site-directed mutagenesis and enzymatic analysis, we demonstrated that the D393A mutation abolished the glucuronidation activity of UGT1A10 toward all substrates. The effects of the alanine mutation at Q(394),D(396), and K(399) on glucuronidation activities were substrate-dependent. Previously, we examined the importance of these residues in UGT2B7. Although D(393) (D(398) in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q(394) (Q(399) in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate binding may explain these differences between UGT1A10 and UGT2B7. We thus postulate that D(393) is critical for the binding of glucuronic acid and that proximal residues, e.g., Q(394) (Q(399) in UGT2B7), play a subtle role in cosubstrate binding in UGT1A10 and UGT2B7. Hence, this study provides important new information needed for the identification and understanding of the binding sites of UGTs, a major step forward in elucidating their molecular mechanism.
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Yan
Y
Xiong
Anne-Sisko
AS
Patana
Michael J
MJ
Miley
Agnieszka K
AK
Zielinska
Stacie M
SM
Bratton
Grover P
GP
Miller
Adrian
A
Goldman
Moshe
M
Finel
Matt R
MR
Redinbo
Anna
A
Radominska-Pandya
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry / Q000378:metabolism*
D007527:Isoenzymes; D011994:Recombinant Proteins; D014535:Uridine Diphosphate Glucuronic Acid; D001224:Aspartic Acid; C476725:bilirubin uridine-diphosphoglucuronosyl transferase 1A10; D014453:Glucuronosyltransferase