Sundaresan Ramya, Ebihara Akio, Kuramitsu Seiki, Yokoyama Shigeyuki, Kumarevel Thirumananseri, Ponnuraj Karthe
Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India.
RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
Biochem Biophys Res Commun. 2015 Sep 18;465(2):174-9. doi: 10.1016/j.bbrc.2015.07.096. Epub 2015 Jul 23.
The enzymatic biosynthesis of L-arginine involves complex, sequential action of many enzymes and ornithine transcarbamylase (OTCase) is one of the essential enzymes in the pathway. In mammals OTCase is part of the urea cycle. Arginine is used in a variety of pharmaceutical and industrial applications and therefore engineering arginine biosynthesis pathway for overproduction of arginine has gained importance. On the other hand, it was found that detrimental mutations in the human OTCase gene resulted clinical hyperammonemia, with subsequent neurological damage. Therefore a better understanding of the structure-function relationship of this enzyme from various sources could be useful for modifying its enzymatic action. Here we report the structure of ornithine transcarbamylase of Thermus thermophilus HB8 (aTtOTCase) at 2.0 Å resolution. On comparison with its homologs, aTtOTCase showed maximum variation at the substrate binding loops namely 80s and SMG/240s loops. The active site geometry of aTtOTCase is unique among its homologs where the side chain of certain residues (Leu57, Arg58 and Arg288) is oriented differently. To study the structural insights of substrate binding in aTtOTCase, docking of carbamoyl phosphate (CP) and ornithine (Orn) was carried out sequentially. Both substrates were unable to bind in a proper orientation in the active site pocket and this could be due to the differently oriented side chains. This suggests that the active site geometry should also undergo fine tuning besides the large structural changes as the enzyme switches from completely open to a substrate bound closed state.
L-精氨酸的酶促生物合成涉及多种酶的复杂、顺序作用，鸟氨酸转氨甲酰酶（OTCase）是该途径中的关键酶之一。在哺乳动物中，OTCase是尿素循环的一部分。精氨酸被用于多种制药和工业应用，因此通过工程改造精氨酸生物合成途径以过量生产精氨酸变得愈发重要。另一方面，人们发现人类OTCase基因中的有害突变会导致临床高氨血症，并随之造成神经损伤。因此，更好地了解来自不同来源的这种酶的结构-功能关系，可能有助于改变其酶促作用。在此，我们报道了嗜热栖热菌HB8的鸟氨酸转氨甲酰酶（aTtOTCase）在2.0 Å分辨率下的结构。与同系物相比，aTtOTCase在底物结合环（即80s环和SMG/240s环）处表现出最大差异。aTtOTCase的活性位点几何结构在其同系物中是独特的，某些残基（Leu57、Arg58和Arg288）的侧链取向不同。为了研究aTtOTCase中底物结合的结构见解，依次进行了氨甲酰磷酸（CP）和鸟氨酸（Orn）的对接。两种底物都无法在活性位点口袋中以合适的取向结合，这可能是由于侧链取向不同所致。这表明，随着酶从完全开放状态转变为底物结合的封闭状态，除了大的结构变化外，活性位点几何结构也应进行微调。
