Watt Gregory, Leoff Christine, Harper April D, Bar-Peled Maor
Complex Carbohydrate Research Center and Department of Plant Biology, University of Georgia, Athens, Georgia 30602-4712, USA.
Plant Physiol. 2004 Apr;134(4):1337-46. doi: 10.1104/pp.103.037192. Epub 2004 Mar 12.
l-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 30 degrees C. The apparent K(m) for NADPH is 90 microm and 16.9 microm for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-beta-l-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.
L-鼠李糖是植物细胞壁果胶多糖、多种次生代谢产物和一些糖蛋白的组成成分。各种鼠李糖基转移酶所利用的鼠李糖的活化核苷酸糖形式的生物合成仍然不清楚，并且尚未纯化出参与其合成的植物酶。相比之下，已在细菌中鉴定出两个基因（rmlC和rmlD），并表明它们编码一种3,5-表异构酶和一种4-酮还原酶，它们共同将dTDP-4-酮-6-脱氧葡萄糖转化为dTDP-β-L-鼠李糖。我们鉴定出一个拟南芥cDNA，其包含与还原酶和表异构酶都具有相似性的结构域。该拟南芥基因编码一种预测分子量约为33.5 kD的蛋白质，在所检测的所有组织中均有转录。在大肠杆菌中表达并纯化的拟南芥蛋白在NADPH存在的情况下将dTDP-4-酮-6-脱氧葡萄糖转化为dTDP-β-L-鼠李糖。这些结果表明，一种单一的植物酶同时具有3,5-表异构酶和4-酮还原酶活性。该酶在30℃、pH 5.5至7.5之间具有最大活性。NADPH的表观K(m)为90微摩尔，dTDP-4-酮-6-脱氧葡萄糖的表观K(m)为16.9微摩尔。拟南芥酶也可以形成UDP-β-L-鼠李糖。据我们所知，这是参与糖核苷酸合成的双功能植物酶的第一个例子，其中单一多肽表现出与两种单独的原核酶相同的活性。
