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Title: Expression, Purification, Assay, and Crystal Structure of Perdeuterated Human Arginase I

Abstract

Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to yield L-ornithine and urea. In order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the X-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase I complexed with 2(S)-amino-6-boronohexanoic acid (ABH) at 1.90 {angstrom} resolution. Prior to the neutron diffraction experiment, it is important to establish that perdeuteration does not cause any unanticipated structural or functional changes. Accordingly, we find that perdeuterated human arginase I exhibits catalytic activity essentially identical to that of the unlabeled enzyme. Additionally, the structure of the perdeuterated human arginase I-ABH complex is identical to that of the corresponding complex with the unlabeled enzyme. Therefore, we conclude that crystals of the perdeuterated human arginase I-ABH complex are suitable for neutron crystallographic study.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930015
Report Number(s):
BNL-80629-2008-JA
Journal ID: ISSN 0003-9861; ABBIA4; TRN: US200822%%1166
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Archives of Biochemistry and Biophysics; Journal Volume: 465; Journal Issue: 1
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ARGINASE; COMPLEXES; CRYSTAL STRUCTURE; CRYSTALS; FUNCTIONALS; HUMAN POPULATIONS; HYDROLYSIS; MANGANESE; NEUTRON DIFFRACTION; NEUTRONS; PURIFICATION; UREA; national synchrotron light source

Citation Formats

Di Costanzo,L., Moulin, M., Haertlein, M., Meilleur, F., and Christianson, D. Expression, Purification, Assay, and Crystal Structure of Perdeuterated Human Arginase I. United States: N. p., 2007. Web. doi:10.1016/j.abb.2007.04.036.
Di Costanzo,L., Moulin, M., Haertlein, M., Meilleur, F., & Christianson, D. Expression, Purification, Assay, and Crystal Structure of Perdeuterated Human Arginase I. United States. doi:10.1016/j.abb.2007.04.036.
Di Costanzo,L., Moulin, M., Haertlein, M., Meilleur, F., and Christianson, D. Mon . "Expression, Purification, Assay, and Crystal Structure of Perdeuterated Human Arginase I". United States. doi:10.1016/j.abb.2007.04.036.
@article{osti_930015,
title = {Expression, Purification, Assay, and Crystal Structure of Perdeuterated Human Arginase I},
author = {Di Costanzo,L. and Moulin, M. and Haertlein, M. and Meilleur, F. and Christianson, D.},
abstractNote = {Arginase is a manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to yield L-ornithine and urea. In order to establish a foundation for future neutron diffraction studies that will provide conclusive structural information regarding proton/deuteron positions in enzyme-inhibitor complexes, we have expressed, purified, assayed, and determined the X-ray crystal structure of perdeuterated (i.e., fully deuterated) human arginase I complexed with 2(S)-amino-6-boronohexanoic acid (ABH) at 1.90 {angstrom} resolution. Prior to the neutron diffraction experiment, it is important to establish that perdeuteration does not cause any unanticipated structural or functional changes. Accordingly, we find that perdeuterated human arginase I exhibits catalytic activity essentially identical to that of the unlabeled enzyme. Additionally, the structure of the perdeuterated human arginase I-ABH complex is identical to that of the corresponding complex with the unlabeled enzyme. Therefore, we conclude that crystals of the perdeuterated human arginase I-ABH complex are suitable for neutron crystallographic study.},
doi = {10.1016/j.abb.2007.04.036},
journal = {Archives of Biochemistry and Biophysics},
number = 1,
volume = 465,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Human arginase I is a potential target for therapeutic intervention in diseases linked to compromised L-arginine homeostasis. Here, we report high-affinity binding of the reaction coordinate analogue inhibitors 2(S)-amino-6-boronohexanoic acid (ABH, Kd = 5 nM) and S-(2-boronoethyl)-L-cysteine (BEC, Kd = 270 nM) to human arginase I, and we report x-ray crystal structures of the respective enzyme-inhibitor complexes at 1.29- and 1.94-Angstrom resolution determined from crystals twinned by hemihedry. The ultrahigh-resolution structure of the human arginase I-ABH complex yields an unprecedented view of the binuclear manganese cluster and illuminates the structural basis for nanomolar affinity: bidentate inner-sphere boronate-manganese coordination interactions andmore » fully saturated hydrogen bond networks with inhibitor {alpha}-amino and {alpha}-carboxylate groups. These interactions are therefore implicated in the stabilization of the transition state for L-arginine hydrolysis. Electron density maps also reveal that active-site residue H141 is protonated as the imidazolium cation. The location of H141 is such that it could function as a general acid to protonate the leaving amino group of L-ornithine during catalysis, and this is a revised mechanistic proposal for arginase. This work serves as a foundation for studying the structural and chemical biology of arginase I in the immune response, and we demonstrate the inhibition of arginase activity by ABH in human and murine myeloid cells.« less
  • The crystal structure of the human arginase I-thiosemicarbazide complex reveals an unusual thiocarbonyl {mu}-sulfide ligand in the binuclear manganese cluster. The CS moiety of thiosemicarbazide bridges Mn2+A and Mn2+B with coordination distances of 2.6 and 2.4 Angstroms, respectively. Otherwise, the binding of thiosemicarbazide to human arginase I does not cause any significant structural changes in the active site. The crystal structure of the unliganded enzyme reveals a hydrogen-bonded water molecule that could support proton transfer between a {mu}-water molecule and H141 to regenerate the nucleophilic {mu}-hydroxide ion in the final step of catalysis.
  • The synthesis of (2S)-2-amino-7,8-epoxyoctanoic acid is reported along with the X-ray crystal structure of its complex with human arginase I, revealing unique coordination interactions with two manganese ions in the enzyme active site.