Synthesis of a New Photoaffinity Probe, 5‐Azido‐[³²P]UDPxylose, by UDPglucuronate Carboxylyase from Wheat Germ

Abstract

The enzyme, UDPglucuronic acid carboxylase (EC 4.1.1.35), was extensively purified from wheat germ, and was used to convert 5-azido-[32P]UDPglucuronic acid to 5-azido-[32P]UDPxylose, for use as a new photoaffinity probe. The carboxylyase was purified approximately 1200-fold using conventional methods, and the enzyme preparation, at the final stage of purification, was stable to storage at -20 degrees C for at least 9 months with little or no loss of activity. The partially purified carboxylyase catalyzed the conversion of 5-azido-[32P]UDPglucuronic acid to 5-azido-[32P]UDPxylose in good yield, and the UDPxylose probe was purified by ion-exchange chromatography, and characterized. The newly synthesized photoaffinity analog, 5-azido-[32P]UDPxylose, should be a valuable tool in the purification of various xylosyltransferases.

Characterisation and expression of the pathway from UDP-glucose to UDP-xylose in differentiating tobacco tissue

“…This conclusion has been based on multiple activity peaks during chromatographic separations but the polypeptides were not purified to homogeneity (Ankel et al, 1967;John et al, 1977;Matern and Grisebach, 1977;Kyossev et al, 1995). More recently the tobacco UDP-glucuronate decarboxylase was purified to apparent homogeneity and its activity could be resolved as distinct M r 87 and 40 kDa soluble polypeptides (Wheatley, 2001;Wheatley et al, 2002).…”

Cell wall deposition and metabolism in wheat grain

“…UDP-D-glucuronic acid can be converted to UDP-D-xylose by UDP-D-glucuronic acid decarboxylase (or UDPxylose synthase UXS). A UXS was purified from wheat germ (Kyossev et al, 1995). Plants have several UXS genes, some are cytosolic while others are membrane-bound located in the Golgi.…”

Nucleoside Diphosphate Sugar Analogues that Target Glycosyltransferases

“…[46] [b-32 P]-5-Azido-UDPÀGlcA (52)w as prepared from compound 51 by using UDPÀGlc 6-dehydrogenase EC 1.1.1.22 [47] and furtherc onverted into [b-32 P]-5-azido-UDPÀ xylose (53)b yt he action of UDPÀglucuronic acid carboxylyase EC 4.1.1.35. [48] As an example, compound 51 wase mployedf or photolabeling glycogenin, an autoglucosyltransferase that initi- ated the de novo biosynthesis of glycogen [49] and( 1,3)-bglucan synthase from Saccharomycesc erevisiae. [50] Compound 52 was also employedt op hotolabel variousGTs , among which is UDPÀglucuronosyltransferase (UGT), am ember of af amily of membrane-bound isoenzymes (UGTs) that catalyze the conjugation of glucuronic acidw ith various compounds of both exogenous and endogenouso rigin, [51] and the streptococcalh yaluronic acid synthase complext hat is responsible for the synthesis of hyaluronic acid.…”