Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry

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• 作者：Guillaumie ; Fanny ; Sterling ; Jason D. ; Jensen ; Knud J. ; Thomas ; Owen R.T. ; Mohnen ; Debra
• 关键词：Biosynthesis ; Enzymatic glycosylation ; Galacturonosyltransferases ; Glycosyltransferases ; Pectin ; Solid-phase ; Aoa ; Aminooxy acetyl ; Boc ; tert-Butyloxycarbonyl ; BSA ; Bovine serum albumin ; DMF ; Dimethylformamide ; DP ; Degree of polymerization ; DTT ; Dithiothr
• 刊名：Carbohydrate Research
• 出版年：2003
• 出版时间：September 10, 2003
• 年：2003
• 卷：338
• 期：19
• 页码：1951-1960
• 全文大小：339 K

文摘

Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through a disulfide-containing cleavable linker, were prepared. The OGAs (degrees of polymerization of 13 and 14) were efficiently immobilized through the reducing end via formation of an oxime linkage. These OGA-derivatized matrices were subsequently employed in novel solid-phase enzymatic reactions, with the pectin biosynthetic enzyme, α-1,4-galacturonosyltransferase, GalAT (solubilized from Arabidopsis thaliana) and the glycosyl donor, uridine diphosphate-galacturonic acid (UDP-GalA). Solid-supported biosynthesis was followed by cleavage of the immobilized OGAs and direct analysis of the products released into the liquid phases by MALDI-TOF mass spectrometry. In time course studies conducted with an immobilized (α-d-GalA)₁₄ and limiting amounts of the glycosyl donor, the predominant product was an OGA extended by one GalA residue at the non-reducing end (i.e., (GalA)₁₅). When UDP-GalA was added in excess compared to immobilized (GalA)₁₃, OGAs up to the 16-mer were synthesized, confirming the non-processivity of the GalAT in vitro.