Hong Lin, Zhao Zongbao, Melançon Charles E, Zhang Hua, Liu Hung-wen
Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712, USA.
J Am Chem Soc. 2008 Apr 9;130(14):4954-67. doi: 10.1021/ja0771383. Epub 2008 Mar 18.
Forosamine (4-dimethylamino)-2,3,4,6-tetradeoxy-beta-D-threo-hexopyranose) is a highly deoxygenated sugar component of several important natural products, including the potent yet environmentally benign insecticide spinosyns. To study D-forosamine biosynthesis, the five genes (spnO, N, Q, R, and S) from the spinosyn gene cluster thought to be involved in the conversion of TDP-4-keto-6-deoxy-D-glucose to TDP-D-forosamine were cloned and heterologously expressed, and the corresponding proteins were purified and their activities examined in vitro. Previous work demonstrated that SpnQ functions as a pyridoxamine 5'-monophosphate (PMP)-dependent 3-dehydrase which, in the presence of the cellular reductase pairs ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase, catalyzes C-3 deoxygenation of TDP-4-keto-2,6-dideoxy-D-glucose. It was also established that SpnR functions as a transaminase which converts the SpnQ product, TDP-4-keto-2,3,6-trideoxy-D-glucose, to TDP-4-amino-2,3,4,6-tetradeoxy-D-glucose. The results presented here provide a full account of the characterization of SpnR and SpnQ and reveal that SpnO and SpnN functions as a 2,3-dehydrase and a 3-ketoreductase, respectively. These two enzymes act sequentially to catalyze C-2 deoxygenation of TDP-4-keto-6-deoxy-D-glucose to form the SpnQ substrate, TDP-4-keto-2,6-dideoxy-D-glucose. Evidence has also been obtained to show that SpnS functions as the 4-dimethyltransferase that converts the SpnR product to TDP-D-forosamine. Thus, the biochemical functions of the five enzymes involved in TDP-D-forosamine formation have now been fully elucidated. The steady-state kinetic parameters for the SpnQ-catalyzed reaction have been determined, and the substrate specificities of SpnQ and SpnR have been explored. The implications of this work for natural product glycodiversification and comparative mechanistic analysis of SpnQ and related NDP-sugar 3-dehydrases E1 and ColD are discussed.
福罗胺（4 - 二甲氨基）-2,3,4,6 - 四脱氧 -β-D-苏式己吡喃糖）是几种重要天然产物中的一种高度脱氧糖成分，包括强效且对环境友好的杀虫剂多杀菌素。为了研究D-福罗胺的生物合成，克隆并异源表达了多杀菌素基因簇中被认为参与将TDP - 4 - 酮 - 6 - 脱氧 - D - 葡萄糖转化为TDP - D - 福罗胺的五个基因（spnO、N、Q、R和S），纯化了相应的蛋白质并在体外检测了它们的活性。先前的研究表明，SpnQ作为一种依赖磷酸吡哆胺5'-单磷酸（PMP）的3 - 脱水酶，在细胞还原酶对铁氧化还原蛋白/铁氧化还原蛋白还原酶或黄素氧还蛋白/黄素氧还蛋白还原酶存在的情况下，催化TDP - 4 - 酮 - 2,6 - 二脱氧 - D - 葡萄糖的C - 3脱氧反应。还确定SpnR作为一种转氨酶，将SpnQ的产物TDP - 4 - 酮 - 2,3,6 - 三脱氧 - D - 葡萄糖转化为TDP - 4 - 氨基 - 2,3,4,6 - 四脱氧 - D - 葡萄糖。此处给出的结果全面描述了SpnR和SpnQ的特性，并揭示SpnO和SpnN分别作为2,3 - 脱水酶和3 - 酮还原酶发挥作用。这两种酶依次作用，催化TDP - 4 - 酮 - 6 - 脱氧 - D - 葡萄糖的C - 2脱氧反应，形成SpnQ的底物TDP - 4 - 酮 - 2,6 - 二脱氧 - D - 葡萄糖。也已获得证据表明SpnS作为将SpnR的产物转化为TDP - D - 福罗胺的4 - 二甲基转移酶发挥作用。因此，现在已经完全阐明了参与TDP - D - 福罗胺形成的五种酶的生化功能。已确定SpnQ催化反应的稳态动力学参数，并探索了SpnQ和SpnR的底物特异性。讨论了这项工作对天然产物糖多样化的意义以及对SpnQ与相关NDP - 糖3 - 脱水酶E1和ColD的比较机制分析。
