Wald Flavia A, Kissen Ralph, du Jardin Patrick, Moreno Silvia
Plant Biochemistry Laboratory, Instituto Leloir (formerly: Instituto de Investigaciones Bioquímicas Fundación Campomar), I.I.B.B.A.-CONICET, Av. Patricias Argentinas 435, (1405) Buenos Aires, Argentina.
Plant Mol Biol. 2003 Jul;52(4):705-14. doi: 10.1023/a:1025061324856.
Many plant autocatalytic glycosyltransferases are implicated in plant polysaccharide biosynthesis. Cloning of cDNAs encoding potato (Solanum tuberosum L.) UDP-Glc:protein transglucosylase (UPTG, EC 2.4.1.112) and expression of the cDNA clone E11 in Escherichia coli have been previously reported. Here, we studied the functional expression of a second cDNA of the enzyme (E2 clone). Northern blots analysis, with specific cDNA probes for the two UPTG isoforms, showed a differential expression pattern of mRNA levels in different potato tissues. Moreover, both UPTG recombinant enzymes showed different kinetic parameters. The recombinant protein encoded by E2 clone has an apparent Imax for UDP-Xyl and UDP-Gal, significantly higher than for UDP-Glc. The Km values for UDP-Glc were 0.45-0.71 microM and the values for UDP-Xyl and UDP-Gal were slightly higher than that of the UDP-Glc (1.2-2.71 microM) for both UPTG recombinant enzymes. The present study revealed further evidence for the proposed role of UPTG in the synthesis of cell wall polysaccharide. It was found a correlation between UPTG transcript levels and the growing state of the tissues in which there was an active synthesis of cell wall components. Southern blot analysis indicates that at least three genes encoding UPTG are present in potato genome. Phylogenetic analysis of both UPTG recombinant proteins showed that they are members of the RGP subfamilies from dicots.
许多植物自身催化的糖基转移酶参与植物多糖的生物合成。此前已有报道克隆编码马铃薯（Solanum tuberosum L.）UDP-葡萄糖：蛋白质转葡糖基酶（UPTG，EC 2.4.1.112）的cDNA，并在大肠杆菌中表达该cDNA克隆E11。在此，我们研究了该酶的第二个cDNA（E2克隆）的功能表达。用针对两种UPTG同工型的特异性cDNA探针进行的Northern印迹分析表明，不同马铃薯组织中mRNA水平存在差异表达模式。此外，两种UPTG重组酶表现出不同的动力学参数。E2克隆编码的重组蛋白对UDP-木糖和UDP-半乳糖的表观最大反应速度明显高于对UDP-葡萄糖的。两种UPTG重组酶对UDP-葡萄糖的Km值为0.45 - 0.71微摩尔，对UDP-木糖和UDP-半乳糖的值略高于UDP-葡萄糖（1.2 - 2.71微摩尔）。本研究揭示了更多证据支持UPTG在细胞壁多糖合成中的作用。发现UPTG转录水平与细胞壁成分活跃合成的组织生长状态之间存在相关性。Southern印迹分析表明马铃薯基因组中至少存在三个编码UPTG的基因。对两种UPTG重组蛋白的系统发育分析表明它们是双子叶植物RGP亚家族的成员。
