title: Crystal structure analysis of ornithine transcarbamylase from Thermus thermophilus --HB8 provides insights on the plasticity of the active site.
abstract: 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.
authors.affiliation: Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India.
authors.forename: Ramya
authors.initials: R
authors.lastname: Sundaresan
authors.affiliation: RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Ebihara
authors.affiliation: RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.affiliation: RIKEN Structural Biology Laboratory, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan; RIKEN Structural Biology Laboratory, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan. Electronic address: kumarevel.thirumananseri@riken.jp.
authors.forename: Thirumananseri
authors.initials: T
authors.lastname: Kumarevel
authors.affiliation: Centre of Advanced Study in Crystallography and Biophysics, University of Madras, Guindy Campus, Chennai 600 025, India. Electronic address: karthe@unom.ac.in.
authors.forename: Karthe
authors.initials: K
authors.lastname: Ponnuraj
mesh_terms: D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002221:Carbamyl Phosphate / Q000737:chemistry*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D009952:Ornithine / Q000737:chemistry*; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002221:Carbamyl Phosphate; D009952:Ornithine; D009954:Ornithine Carbamoyltransferase
keywords: Active site plasticity; Apo structure; Arginine biosynthesis pathway; OTCase; Thermus thermophilus HB8