Johansson E, Steffens J J, Lindqvist Y, Schneider G
Department of Medical Biochemistry and Biophysics Karolinska Institutet S-171 77, Stockholm, Sweden.
Structure. 2000 Oct 15;8(10):1037-47. doi: 10.1016/s0969-2126(00)00512-8.
The biosynthesis of the essential amino acid lysine in higher fungi and cyanobacteria occurs via the alpha-aminoadipate pathway, which is completely different from the lysine biosynthetic pathway found in plants and bacteria. The penultimate reaction in the alpha-aminoadipate pathway is catalysed by NADPH-dependent saccharopine reductase. We set out to determine the structure of this enzyme as a first step in exploring the structural biology of fungal lysine biosynthesis.
We have determined the three-dimensional structure of saccharopine reductase from the plant pathogen Magnaporthe grisea in its apo form to 2.0 A resolution and as a ternary complex with NADPH and saccharopine to 2.1 A resolution. Saccharopine reductase is a homodimer, and each subunit consists of three domains, which are not consecutive in amino acid sequence. Domain I contains a variant of the Rossmann fold that binds NADPH. Domain II folds into a mixed seven-stranded beta sheet flanked by alpha helices and is involved in substrate binding and dimer formation. Domain III is all-helical. The structure analysis of the ternary complex reveals a large movement of domain III upon ligand binding. The active site is positioned in a cleft between the NADPH-binding domain and the second alpha/beta domain. Saccharopine is tightly bound to the enzyme via a number of hydrogen bonds to invariant amino acid residues.
On the basis of the structure of the ternary complex of saccharopine reductase, an enzymatic mechanism is proposed that includes the formation of a Schiff base as a key intermediate. Despite the lack of overall sequence homology, the fold of saccharopine reductase is similar to that observed in some enzymes of the diaminopimelate pathway of lysine biosynthesis in bacteria. These structural similarities suggest an evolutionary relationship between two different major families of amino acid biosynthetic pathway, the glutamate and aspartate families.
高等真菌和蓝细菌中必需氨基酸赖氨酸的生物合成通过α-氨基己二酸途径进行，这与植物和细菌中的赖氨酸生物合成途径完全不同。α-氨基己二酸途径中的倒数第二个反应由NADPH依赖性的酵母氨酸还原酶催化。我们着手确定该酶的结构，作为探索真菌赖氨酸生物合成结构生物学的第一步。
我们已经确定了植物病原体稻瘟病菌中酵母氨酸还原酶的三维结构，其无配体形式的分辨率为2.0 Å，与NADPH和酵母氨酸形成的三元复合物的分辨率为2.1 Å。酵母氨酸还原酶是一种同型二聚体，每个亚基由三个结构域组成，这些结构域在氨基酸序列中不连续。结构域I包含结合NADPH的Rossmann折叠变体。结构域II折叠成一个由α螺旋侧翼的混合七链β折叠片，参与底物结合和二聚体形成。结构域III全是α螺旋。三元复合物的结构分析表明，配体结合后结构域III发生了较大的移动。活性位点位于NADPH结合结构域和第二个α/β结构域之间的裂隙中。酵母氨酸通过与不变氨基酸残基形成的多个氢键与酶紧密结合。
基于酵母氨酸还原酶三元复合物的结构，提出了一种酶促机制，其中包括形成席夫碱作为关键中间体。尽管缺乏整体序列同源性，但酵母氨酸还原酶的折叠与细菌中赖氨酸生物合成二氨基庚二酸途径的一些酶中观察到的折叠相似。这些结构相似性表明了氨基酸生物合成途径的两个不同主要家族，即谷氨酸家族和天冬氨酸家族之间的进化关系。
