Importance of Gly-13 for the coenzyme binding of human UDP-glucose dehydrogenase.

The Journal of Biological Chemistry
Jae-Wan HuhSung-Woo Cho

Abstract

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 i...Continue Reading

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Citations

Oct 11, 2011·PloS One·Venkatachalam RajakannanRobert C Robinson
Mar 19, 2013·Environmental Toxicology and Pharmacology·Sharad VermaAbha Mishra
Oct 12, 2007·Journal of Biochemistry and Molecular Biology·Jae-Wan HuhSung-Woo Cho
Feb 9, 2012·Biochimica Et Biophysica Acta·Sohee PharkDonggeun Sul
Dec 29, 2006·International Journal of Cancer. Journal International Du Cancer·Waldemar WaldeckKlaus Braun

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