Crystal Structure of Novel Dye-linked L-Proline Dehydrogenase from Hyperthermophilic Archaeon Aeropyrum pernix
Crystal Structure of Novel Dye-linked L-Proline Dehydrogenase from Hyperthermophilic Archaeon Aeropyrum pernix
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DOI：
10.1074/jbc.m111.319038
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发表时间：
2012-06-08
影响因子：
4.8
通讯作者：
Ohshima, Toshihisa
中科院分区：
文献类型：
--
作者：
Sakuraba, Haruhiko;Satomura, Takenori;Ohshima, Toshihisa
迄今为止，已在超嗜热古菌中鉴定出两种类型的染料连接的L-脯氨酸脱氢酶（PDH 1，α 4 β 4型异源八聚体，和PDH 2，α β γ δ型异源四聚体）。在这里，我们报告的晶体结构的第三种类型的L-脯氨酸脱氢酶，发现在有氧超嗜热古菌Aeropyrum pernix，其结构（同源二聚体）是比以前研究的L-脯氨酸脱氢酶简单得多。该结构在1.92埃的分辨率下确定。不对称单元包含一个亚基，并且晶体学2倍轴产生功能性二聚体。亚基的整体折叠显示与负责催化L-脯氨酸脱氢的PDH 1 β-亚基的折叠相似。然而，在亚基-亚基界面的情况下，A。pernix酶与PDH 1完全不同。在A. Pernix酶有助于独特的二聚体缔合。此外，由从FAD结合结构域延伸的尾部提供的C-末端Leu（428）屏蔽了活性位点，并且L-脯氨酸分子被截留在活性位点空腔内。Leu（428）缺失突变体对L-脯氨酸的Km值比野生型酶的Km值大约800倍，尽管两种酶之间的k（cat）值差异不大。这表明C-末端Leu（428）不直接参与催化，但它对于维持对底物的高亲和力是必不可少的。这是第一次描述与L-脯氨酸结合的LPDH结构，它提供了新的洞察LPDH的底物结合。
Two types of dye-linked L-proline dehydrogenase (PDH1, alpha 4 beta 4-type hetero-octamer, and PDH2, alpha beta gamma delta-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of L-proline dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied L-proline dehydrogenases. The structure was determined at a resolution of 1.92 angstrom. The asymmetric unit contained one subunit, and a crystallographic 2-fold axis generated the functional dimer. The overall fold of the subunit showed similarity to that of the PDH1 beta-subunit, which is responsible for catalyzing L-proline dehydrogenation. However, the situation at the subunit-subunit interface of the A. pernix enzyme was totally different from that in PDH1. The presence of additional surface elements in the A. pernix enzyme contributes to a unique dimer association. Moreover, the C-terminal Leu(428), which is provided by a tail extending from the FAD-binding domain, shielded the active site, and an L-proline molecule was entrapped within the active site cavity. The K-m value of a Leu(428) deletion mutant for L-proline was about 800 times larger than the K-m value of the wild-type enzyme, although the k(cat) values did not differ much between the two enzymes. This suggests the C-terminal Leu(428) is not directly involved in catalysis, but it is essential for maintaining a high affinity for the substrate. This is the first description of an LPDH structure with L-proline bound, and it provides new insight into the substrate binding of LPDH.