Importance of Gly-13 for the coenzyme binding of human UDP-glucose dehydrogenase.
UDP-glucose dehydrogenase (UGDH) is the unique pathway enzyme furnishing in vertebrates UDP-glucuronate for numerous transferases. In this report, we have identified an NAD(+)-binding site within human UGDH by photoaffinity labeling with a specific probe, [(32)P]nicotinamide 2-azidoadenosine dinucleotide (2N(3) NAD(+)), and cassette mutagenesis. For this work, we have chemically synthesized a 1509-base pair gene encoding human UGDH and expressed it in Escherichia coli as a soluble protein. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+)-binding site. Amino acid sequencing and compositional analysis identified the NAD(+)-binding site of UGDH as the region containing the sequence ICCIGAXYVGGPT, corresponding to Ile-7 through Thr-19 of the amino acid sequence of human UGDH. The unidentified residue, X, can be designated as a photolabeled Gly-13 because the sequences including the glycine residue in question have a complete identity with those of other UGDH species known. The importance of Gly-13 residue in the binding of NAD(+) was further examined with a G13E mutant by cassette mutagenesis. The mutagenesis at Gly-13 had no effects on the expression or stability of the mutant. Enzyme activity of the G13E point mutant was not measurable under normal assay conditions, suggesting an important role for the Gly-13 residue. No incorporation of [(32)P]2N(3)NAD(+) was observed for the G13E mutant. These results indicate that Gly-13 plays an important role for efficient binding of NAD(+) to human UGDH.
Huh
Jae-Wan
JW
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul 138-736, Korea.
Yoon
Hye-Young
HY
Lee
Hyun-Ju
HJ
Choi
Won-Beom
WB
Yang
Seung-Ju
SJ
Cho
Sung-Woo
SW
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D009243:NAD; D004247:DNA; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014357:Trypsin; D005998:Glycine