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Title: The Catalytic Pocket of the Ring-hydroxylating Dioxygenase from Sphingomonas CHY-1

Abstract

No abstract prepared.

Authors:
; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930176
Report Number(s):
BNL-80837-2008-JA
Journal ID: ISSN 0006-291X; BBRCA9; TRN: US200822%%1221
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 352
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; OXYGENASES; CATALYTIC EFFECTS; MICROORGANISMS; HYDROXYLATION; national synchrotron light source

Citation Formats

Jakoncic,J., Jouanneau, Y., Meyer, C., and Stojanoff, V. The Catalytic Pocket of the Ring-hydroxylating Dioxygenase from Sphingomonas CHY-1. United States: N. p., 2006. Web.
Jakoncic,J., Jouanneau, Y., Meyer, C., & Stojanoff, V. The Catalytic Pocket of the Ring-hydroxylating Dioxygenase from Sphingomonas CHY-1. United States.
Jakoncic,J., Jouanneau, Y., Meyer, C., and Stojanoff, V. Sun . "The Catalytic Pocket of the Ring-hydroxylating Dioxygenase from Sphingomonas CHY-1". United States. doi:.
@article{osti_930176,
title = {The Catalytic Pocket of the Ring-hydroxylating Dioxygenase from Sphingomonas CHY-1},
author = {Jakoncic,J. and Jouanneau, Y. and Meyer, C. and Stojanoff, V.},
abstractNote = {No abstract prepared.},
doi = {},
journal = {Biochemical and Biophysical Research Communications},
number = ,
volume = 352,
place = {United States},
year = {Sun Jan 01 00:00:00 EST 2006},
month = {Sun Jan 01 00:00:00 EST 2006}
}
  • The ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 {angstrom} by molecular replacement revealed the enzyme to share the same global {alpha}{sub 3}{beta}{sub 3} structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate bindingmore » pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 {angstrom}. An 11 {angstrom} long water channel extends from the interface between the {alpha} and {beta} subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.« less
  • We present the structure of LinB, a 33-kDa haloalkane dehalogenase from Sphingomonas paucimobilis UT26, at 0.95 {angstrom} resolution. The data have allowed us to directly observe the anisotropic motions of the catalytic residues. In particular, the side-chain of the catalytic nucleophile, Asp108, displays a high degree of disorder. It has been modeled in two conformations, one similar to that observed previously (conformation A) and one strained (conformation B) that approached the catalytic base (His272). The strain in conformation B was mainly in the C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle (126{sup o}) that deviated by 13.4{sup o} from the 'ideal' bond anglemore » of 112.6{sup o}. On the basis of these observations, we propose a role for the charge state of the catalytic histidine in determining the geometry of the catalytic residues. We hypothesized that double-protonation of the catalytic base (His272) reduces the distance between the side-chain of this residue and that of the Asp108. The results of molecular dynamics simulations were consistent with the structural data showing that protonation of the His272 side-chain nitrogen atoms does indeed reduce the distance between the side-chains of the residues in question, although the simulations failed to demonstrate the same degree of strain in the Asp108 C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle. Instead, the changes in the molecular dynamics structures were distributed over several bond and dihedral angles. Quantum mechanics calculations on LinB with 1-chloro-2,2-dimethylpropane as a substrate were performed to determine which active site conformations and protonation states were most likely to result in catalysis. It was shown that His272 singly protonated at N{sub {delta}1} and Asp108 in conformation A gave the most exothermic reaction ({Delta}H = -22 kcal/mol). With His272 doubly protonated at N{sub {delta}1} and N{sub {epsilon}2}, the reactions were only slightly exothermic or were endothermic. In all calculations starting with Asp108 in conformation B, the Asp108 C{sub {alpha}}-C{sub {beta}}-C{sub {gamma}} angle changed during the reaction and the Asp108 moved to conformation A. The results presented here indicate that the positions of the catalytic residues and charge state of the catalytic base are important for determining reaction energetics in LinB.« less
  • Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-Hydroxylating Monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met314 and two His residues as ligands. Mutation of Met314 to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met314 as amore » CuM ligand, we determined the structure of the M314I-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The M314I-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the M314I-PHM crystal form and showed that it does not differ from the structure of wt-PHM in the original crystals. M314I-PHM was also shown to be less stable than wt-PHM by Differential Scanning Calorimetry (DSC). Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.« less