Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site
Abstract
Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure–activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Furthermore, our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC); National Inst. of Health; Welch Foundation
- OSTI Identifier:
- 1346210
- Grant/Contract Number:
- AC02-06CH11357; R01 CA154754; R01 GM 104896; F-1778
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Biochemistry
- Additional Journal Information:
- Journal Volume: 56; Journal Issue: 6; Journal ID: ISSN 0006-2960
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; crystals; salts; peptides and proteins; monomers; ionic strength
Citation Formats
Yan, Wupeng, Stone, Everett, and Zhang, Yan Jessie. Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site. United States: N. p., 2017.
Web. doi:10.1021/acs.biochem.6b01172.
Yan, Wupeng, Stone, Everett, & Zhang, Yan Jessie. Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site. United States. https://doi.org/10.1021/acs.biochem.6b01172
Yan, Wupeng, Stone, Everett, and Zhang, Yan Jessie. Fri .
"Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site". United States. https://doi.org/10.1021/acs.biochem.6b01172. https://www.osti.gov/servlets/purl/1346210.
@article{osti_1346210,
title = {Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site},
author = {Yan, Wupeng and Stone, Everett and Zhang, Yan Jessie},
abstractNote = {Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure–activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Furthermore, our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.},
doi = {10.1021/acs.biochem.6b01172},
journal = {Biochemistry},
number = 6,
volume = 56,
place = {United States},
year = {Fri Jan 20 00:00:00 EST 2017},
month = {Fri Jan 20 00:00:00 EST 2017}
}
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Works referenced in this record:
The Effect of Replacement of Methionine by Homocystine on Survival of Malignant and Normal Adult Mammalian Cells in Culture
journal, April 1974
- Halpern, B. C.; Clark, B. R.; Hardy, D. N.
- Proceedings of the National Academy of Sciences, Vol. 71, Issue 4
Methionine Dependency of Malignant Tumors: A Possible Approach for Therapy
journal, October 1990
- Breillout, F.; Antoine, E.; Poupon, M. F.
- JNCI: Journal of the National Cancer Institute, Vol. 82, Issue 20
Evaluation of brain tumors using dynamic 11C-methionine-PET
journal, April 2012
- Aki, Tatsuki; Nakayama, Noriyuki; Yonezawa, Shingo
- Journal of Neuro-Oncology, Vol. 109, Issue 1
Three-dimensional Gaussian model to define brain metastasis limits on 11C-methionine PET
journal, December 2008
- Tang, Bich-Ngoc-Thanh; Van Simaeys, Gaetan; Devriendt, Daniel
- Radiotherapy and Oncology, Vol. 89, Issue 3
[11C]-l-Methionine positron emission tomography in the management of children and young adults with brain tumors
journal, July 2009
- Galldiks, Norbert; Kracht, Lutz W.; Berthold, Frank
- Journal of Neuro-Oncology, Vol. 96, Issue 2
Polyamine metabolism and cancer
journal, April 2003
- Thomas, Thresia; Thomas, T. J.
- Journal of Cellular and Molecular Medicine, Vol. 7, Issue 2
Ornithine decarboxylase activity is critical for cell transformation
journal, November 1992
- Auvinen, Merja; Paasinen, Aino; Andersson, Leif C.
- Nature, Vol. 360, Issue 6402
Quantitation of total homocysteine, total cysteine, and methionine in normal serum and urine using capillary gas chromatography-mass spectrometry
journal, April 1987
- Stabler, Sally P.; Marcell, Paul D.; Podell, Elaine R.
- Analytical Biochemistry, Vol. 162, Issue 1
Regulation of O6-methylguanine-DNA methyltransferase by methionine in human tumour cells
journal, March 1997
- Kokkinakis, Dm; von Wronski, Ma; Vuong, Th
- British Journal of Cancer, Vol. 75, Issue 6
Pharmacokinetics, Methionine Depletion, and Antigenicity of Recombinant Methioninase in Primates
journal, March 2004
- Yang, Zhijian; Wang, Junhua; Yoshioka, Takayuki
- Clinical Cancer Research, Vol. 10, Issue 6
Determinants of Enzymatic Specificity in the Cys-Met-Metabolism PLP-Dependent Enzyme Family: Crystal Structure of Cystathionine γ-Lyase from Yeast and Intrafamiliar Structure Comparison
journal, January 2003
- Messerschmidt, A.; Worbs, M.; Steegborn, C.
- Biological Chemistry, Vol. 384, Issue 3
De Novo Engineering of a Human Cystathionine-γ-Lyase for Systemic l -Methionine Depletion Cancer Therapy
journal, August 2012
- Stone, Everett; Paley, Olga; Hu, Jian
- ACS Chemical Biology, Vol. 7, Issue 11
Ligation-independent cloning of PCR products (LIC-PCR)
journal, January 1990
- Aslanidis, Charalampos; de Jong, Pieter J.
- Nucleic Acids Research, Vol. 18, Issue 20
Overview of the CCP 4 suite and current developments
journal, March 2011
- Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.
- Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
Crystallographic fast Fourier transforms
journal, March 1973
- Ten Eyck, L. F.
- Acta Crystallographica Section A, Vol. 29, Issue 2
A phased translation function
journal, October 1988
- Read, R. J.; Schierbeek, A. J.
- Journal of Applied Crystallography, Vol. 21, Issue 5
Phaser crystallographic software
journal, July 2007
- McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
- Journal of Applied Crystallography, Vol. 40, Issue 4
PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010
- Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
Use of knowledge-based restraints in phenix.refine to improve macromolecular refinement at low resolution
journal, March 2012
- Headd, Jeffrey J.; Echols, Nathaniel; Afonine, Pavel V.
- Acta Crystallographica Section D Biological Crystallography, Vol. 68, Issue 4
Bulk-solvent and overall scaling revisited: faster calculations, improved results
journal, March 2013
- Afonine, P. V.; Grosse-Kunstleve, R. W.; Adams, P. D.
- Acta Crystallographica Section D Biological Crystallography, Vol. 69, Issue 4
Features and development of Coot
journal, March 2010
- Emsley, P.; Lohkamp, B.; Scott, W. G.
- Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
PROCHECK: a program to check the stereochemical quality of protein structures
journal, April 1993
- Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.
- Journal of Applied Crystallography, Vol. 26, Issue 2
MolProbity: all-atom contacts and structure validation for proteins and nucleic acids
journal, May 2007
- Davis, I. W.; Leaver-Fay, A.; Chen, V. B.
- Nucleic Acids Research, Vol. 35, Issue Web Server
Binding of Pyridoxal 5‘-Phosphate to the Heme Protein Human Cystathionine β-Synthase
journal, February 1999
- Kery, Vladimir; Poneleit, Loelle; Meyer, Jeffrey D.
- Biochemistry, Vol. 38, Issue 9
[10] Pyridoxal phosphate-binding site in enzymes: Reduction and comparison of sequences
book, January 1984
- Morino, Yoshimasa; Nagashima, Fujio
- Methods in Enzymology
Methionine γ-lyase: Mechanistic deductions from the kinetic pH-effects
journal, October 2009
- Faleev, Nicolai G.; Alferov, Kirill V.; Tsvetikova, Marina A.
- Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1794, Issue 10
Role of Tyrosine 114 ofL -Methionine γ-lyase fromPseudomonas putida
journal, January 2000
- Inoue, Hiroyuki; Inagaki, Kenji; Adachi, Naoki
- Bioscience, Biotechnology, and Biochemistry, Vol. 64, Issue 11
Purification and Characterization of
l
-Methionine γ-Lyase from
Brevibacterium linens
BL2
journal, September 1998
- Dias, Benjamin; Weimer, Bart
- Applied and Environmental Microbiology, Vol. 64, Issue 9
YtjE from
Lactococcus lactis
IL1403 Is a C-S Lyase with α,γ-Elimination Activity toward Methionine
journal, July 2006
- Martínez-Cuesta, M. Carmen; Peláez, Carmen; Eagles, John
- Applied and Environmental Microbiology, Vol. 72, Issue 7
Cloning and Characterization of Two
Lactobacillus casei
Genes Encoding a Cystathionine Lyase
journal, January 2008
- Irmler, Stefan; Raboud, Sylvie; Beisert, Beata
- Applied and Environmental Microbiology, Vol. 74, Issue 1
X-ray structure of MalY from Escherichia coli: a pyridoxal 5′-phosphate-dependent enzyme acting as a modulator in mal gene expression
journal, March 2000
- Clausen, Tim; Schlegel, Anja; Peist, Ralf
- The EMBO Journal, Vol. 19, Issue 5
Effects of Salts on the Conformation and Catalytic Properties of D-Amino Acid Aminotransferase
journal, May 2002
- Ro, Hyeon-Su
- BMB Reports, Vol. 35, Issue 3
CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity
journal, November 2011
- Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto
- Applied Microbiology and Biotechnology, Vol. 94, Issue 5
PLP undergoes conformational changes during the course of an enzymatic reaction
journal, January 2014
- Ngo, Ho-Phuong-Thuy; Cerqueira, Nuno M. F. S. A.; Kim, Jin-Kwang
- Acta Crystallographica Section D Biological Crystallography, Vol. 70, Issue 2
Works referencing / citing this record:
Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii
journal, July 2018
- Maresi, Elena; Janson, Giacomo; Fruncillo, Silvia
- International Journal of Molecular Sciences, Vol. 19, Issue 7