Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis

Knapp, Madison; Jo, Minshik; Henthorn, Courtney L.; Brimberry, Marley; Gnann, Andrew D.; Dowling, Daniel P.; Bridwell-Rabb, Jennifer

doi:10.1021/acsbiomedchemau.4c00089

Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis

Journal Article · Wed Nov 20 00:00:00 EST 2024 · ACS Bio & Med Chem Au

DOI:https://doi.org/10.1021/acsbiomedchemau.4c00089· OSTI ID:2482614

^[1]; Jo, Minshik ^[1]; Henthorn, Courtney L. ^[1]; Brimberry, Marley ^[1]; Gnann, Andrew D. ^[2]; Dowling, Daniel P. ^[2]; ^[1]

University of Michigan, Ann Arbor, MI (United States)
University of Massachusetts, Boston, MA (United States)

Chlorophyll (Chl) is one of Nature’s most complex pigments to biosynthesize and derivatize. This pigment is vital for survival and also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production of Chl, organisms also invest in mechanisms to control its degradation and recycling. One important enzyme that is involved in these latter processes is chlorophyllase. This enzyme is employed by numerous photosynthetic organisms to hydrolyze the phytol tail of Chl. Although traditionally thought to catalyze the first step of Chl degradation, recent work suggests that chlorophyllase is instead employed during times of abiotic stress or conditions that produce reactive oxygen species. However, the molecular details regarding how chlorophyllases are regulated to function under such conditions remain enigmatic. Here, we investigate the Arabidopsis thaliana chlorophyllase isoform AtCLH2 using site-directed mutagenesis, mass spectrometry, dynamic light scattering, size-exclusion multiangle light scattering, and both steady-state enzyme kinetic and thermal stability measurements. Through these experiments, we show that AtCLH2 exists as a monomer in solution and contains two disulfide bonds. One disulfide bond putatively maps to the active site, whereas the other links two N-terminal Cys residues together. These disulfide bonds are cleaved by chemical or chemical and protein-based reductants, respectively, and are integral to maintaining the activity, stability, and substrate scope of the enzyme. This work suggests that Cys residue oxidation in chlorophyllases is an emerging regulatory strategy for controlling the hydrolysis of Chl pigments.

View Journal Article

Research Organization:: Univ. of Michigan, Ann Arbor, MI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: SC0021240

OSTI ID:: 2482614

Alternate ID(s):: OSTI ID: 2482147

Journal Information:: ACS Bio & Med Chem Au, Vol. 4, Issue 6; ISSN 2694-2437

Publisher:: ACS PublicationsCopyright Statement

Country of Publication:: United States

Language:: English

References (65)

The green pigment of life Knapp, Madison; Bridwell-Rabb, Jennifer Nature Chemistry, Vol. 14, Issue 10 https://doi.org/10.1038/s41557-022-01052-6	journal	September 2022
The Degradation of Chlorophyll — a Biological Enigma Hendry, George A. F.; Houghton, Jennifer D.; Brown, Stanley B. New Phytologist, Vol. 107, Issue 2 https://doi.org/10.1111/j.1469-8137.1987.tb00181.x	journal	October 1987
Chlorophyll dephytylation in chlorophyll metabolism: a simple reaction catalyzed by various enzymes Lin, Yao-Pin; Charng, Yee-yung Plant Science, Vol. 302 https://doi.org/10.1016/j.plantsci.2020.110682	journal	January 2021
Chlorophyll metabolism Tanaka, Ayumi; Tanaka, Ryouichi Current Opinion in Plant Biology, Vol. 9, Issue 3 https://doi.org/10.1016/j.pbi.2006.03.011	journal	June 2006
Pathways of Oxidative Damage Imlay, James A. Annual Review of Microbiology, Vol. 57, Issue 1 https://doi.org/10.1146/annurev.micro.57.030502.090938	journal	October 2003
Oxidative stress in cyanobacteria Latifi, Amel; Ruiz, Marion; Zhang, Cheng-Cai FEMS Microbiology Reviews, Vol. 33, Issue 2 https://doi.org/10.1111/j.1574-6976.2008.00134.x	journal	March 2009
Reactive oxygen species, abiotic stress and stress combination Choudhury, Feroza K.; Rivero, Rosa M.; Blumwald, Eduardo The Plant Journal, Vol. 90, Issue 5 https://doi.org/10.1111/tpj.13299	journal	November 2016
The Significance of Reactive Oxygen Species and Antioxidant Defense System in Plants: A Concise Overview Dumanović, Jelena; Nepovimova, Eugenie; Natić, Maja Frontiers in Plant Science, Vol. 11 https://doi.org/10.3389/fpls.2020.552969	journal	January 2021
Cellular defenses against damage from reactive oxygen species Yu, B. P. Physiological Reviews, Vol. 74, Issue 1 https://doi.org/10.1152/physrev.1994.74.1.139	journal	January 1994
Thioredoxin and Thioredoxin Target Proteins: From Molecular Mechanisms to Functional Significance Lee, Samuel; Kim, Soo Min; Lee, Richard T. Antioxidants & Redox Signaling, Vol. 18, Issue 10 https://doi.org/10.1089/ars.2011.4322	journal	April 2013
Untersuchungen über Chlorophyll. XI. Über Chlorophyllase Willstätter, Richard; Stoll, Arthur Justus Liebig's Annalen der Chemie, Vol. 378, Issue 1 https://doi.org/10.1002/jlac.19113780103	journal	January 1911
The chlorophyllases AtCLH1 and AtCLH2 are not essential for senescence-related chlorophyll breakdown in Arabidopsis thaliana Schenk, Nicole; Schelbert, Silvia; Kanwischer, Marion FEBS Letters, Vol. 581, Issue 28 https://doi.org/10.1016/j.febslet.2007.10.060	journal	November 2007
Subcellular localization of chlorophyllase2 reveals it is not involved in chlorophyll degradation during senescence in Arabidopsis thaliana Hu, Xueyun; Jia, Ting; Hörtensteiner, Stefan Plant Science, Vol. 290 https://doi.org/10.1016/j.plantsci.2019.110314	journal	January 2020
A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds Jo, Minshik; Knapp, Madison; Boggs, David G. Journal of Biological Chemistry, Vol. 299, Issue 3 https://doi.org/10.1016/j.jbc.2023.102958	journal	March 2023
Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores Hu, Xueyun; Makita, Satoru; Schelbert, Silvia Plant Physiology, Vol. 167, Issue 3 https://doi.org/10.1104/pp.114.252023	journal	January 2015
Differential Expression of a Novel Gene in Response to Coronatine, Methyl Jasmonate, and Wounding in the Coi1Mutant of Arabidopsis1 Benedetti, Celso E.; Costa, Cinthia L.; Turcinelli, Silvia R. Plant Physiology, Vol. 116, Issue 3 https://doi.org/10.1104/pp.116.3.1037	journal	March 1998
Chlorophyllase 1, a Damage Control Enzyme, Affects the Balance between Defense Pathways in Plants Kariola, Tarja; Brader, Günter; Li, Jing The Plant Cell, Vol. 17, Issue 1 https://doi.org/10.1105/tpc.104.025817	journal	December 2004
Different Mechanisms Are Responsible for Chlorophyll Dephytylation during Fruit Ripening and Leaf Senescence in Tomato Guyer, Luzia; Hofstetter, Silvia Schelbert; Christ, Bastien Plant Physiology, Vol. 166, Issue 1 https://doi.org/10.1104/pp.114.239541	journal	July 2014
Transgenic broccoli (Brassica oleracea var. italica) with antisense chlorophyllase (BoCLH1) delays postharvest yellowing Chen, Long-Fang O.; Lin, Chin-Hong; Kelkar, Swati M. Plant Science, Vol. 174, Issue 1 https://doi.org/10.1016/j.plantsci.2007.09.006	journal	January 2008
Chlorophyll breakdown by chlorophyllase: isolation and functional expression of the Chlase1 gene from ethylene-treated Citrus fruit and its regulation during development Jacob-Wilk, Debora; Holland, Doron; Goldschmidt, Eliezer E. The Plant Journal, Vol. 20, Issue 6 https://doi.org/10.1046/j.1365-313X.1999.00637.x	journal	December 1999
Dual N- and C-Terminal Processing of Citrus Chlorophyllase Precursor Within the Plastid Membranes leads to the Mature Enzyme Azoulay-Shemer, T.; Harpaz-Saad, S.; Cohen-Peer, R. Plant and Cell Physiology, Vol. 52, Issue 1 https://doi.org/10.1093/pcp/pcq174	journal	November 2010
Arabidopsis CHLOROPHYLLASE 1 protects young leaves from long-term photodamage by facilitating FtsH-mediated D1 degradation in photosystem II repair Tian, Ya-nan; Zhong, Rui-hao; Wei, Jun-bin Molecular Plant, Vol. 14, Issue 7 https://doi.org/10.1016/j.molp.2021.04.006	journal	July 2021
AtCLH2, a Typical but Possibly Distinctive Chlorophyllase Gene in Arabidopsis Liao, Yang; An, Kun; Zhou, Xiao Journal of Integrative Plant Biology, Vol. 49, Issue 4 https://doi.org/10.1111/j.1744-7909.2007.00444.x	journal	April 2007
Mechanism and Significance of Chlorophyll Breakdown Christ, Bastien; Hörtensteiner, Stefan Journal of Plant Growth Regulation, Vol. 33, Issue 1 https://doi.org/10.1007/s00344-013-9392-y	journal	November 2013
The ultra-processed foods hypothesis: a product processed well beyond the basic ingredients in the package Visioli, Francesco; Marangoni, Franca; Fogliano, Vincenzo Nutrition Research Reviews, Vol. 36, Issue 2 https://doi.org/10.1017/S0954422422000117	journal	June 2022
HullRad: Fast Calculations of Folded and Disordered Protein and Nucleic Acid Hydrodynamic Properties Fleming, Patrick J.; Fleming, Karen G. Biophysical Journal, Vol. 114, Issue 4 https://doi.org/10.1016/j.bpj.2018.01.002	journal	February 2018
Inference of Macromolecular Assemblies from Crystalline State Krissinel, Evgeny; Henrick, Kim Journal of Molecular Biology, Vol. 372, Issue 3 https://doi.org/10.1016/j.jmb.2007.05.022	journal	September 2007
ExPASy: the proteomics server for in-depth protein knowledge and analysis Gasteiger, E. Nucleic Acids Research, Vol. 31, Issue 13 https://doi.org/10.1093/nar/gkg563	journal	July 2003
ColabFold: making protein folding accessible to all Mirdita, Milot; Schütze, Konstantin; Moriwaki, Yoshitaka Nature Methods, Vol. 19, Issue 6 https://doi.org/10.1038/s41592-022-01488-1	journal	May 2022
Highly accurate protein structure prediction with AlphaFold Jumper, John; Evans, Richard; Pritzel, Alexander Nature https://doi.org/10.1038/s41586-021-03819-2	journal	July 2021
Mechanistic analysis of wheat chlorophyllase Arkus, Kiani A. J.; Cahoon, Edgar B.; Jez, Joseph M. Archives of Biochemistry and Biophysics, Vol. 438, Issue 2, p. 146-155 https://doi.org/10.1016/j.abb.2005.04.019	journal	June 2005
Crystal structures of two functionally different thioredoxins in spinach chloroplasts 1 1Edited by R. Huber Capitani, Guido; Marković-Housley, Zora; DelVal, Gregoire Journal of Molecular Biology, Vol. 302, Issue 1 https://doi.org/10.1006/jmbi.2000.4006	journal	September 2000
Evolution of the thioredoxin system as a step enabling adaptation to oxidative stress Balsera, Monica; Buchanan, Bob B. Free Radical Biology and Medicine, Vol. 140 https://doi.org/10.1016/j.freeradbiomed.2019.03.003	journal	August 2019
Estimating the number of protein folds and families from complete genome data 1 1Edited by J. Thornton Wolf, Yuri I.; Grishin, Nick V.; Koonin, Eugene V. Journal of Molecular Biology, Vol. 299, Issue 4 https://doi.org/10.1006/jmbi.2000.3786	journal	June 2000
Divergent Evolution of Enzymatic Function: Mechanistically Diverse Superfamilies and Functionally Distinct Suprafamilies Gerlt, John A.; Babbitt, Patricia C. Annual Review of Biochemistry, Vol. 70, Issue 1 https://doi.org/10.1146/annurev.biochem.70.1.209	journal	June 2001
Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT Li, Bin; Jo, Minshik; Liu, Jianxin Communications Biology, Vol. 5, Issue 1 https://doi.org/10.1038/s42003-022-03226-x	journal	March 2022
Protein folds: molecular systematics in three dimensions Zhang, C.; DeLisi*, C. Cellular and Molecular Life Sciences, Vol. 58, Issue 1 https://doi.org/10.1007/PL00000779	journal	January 2001
Evolution of Protein Quaternary Structure in Response to Selective Pressure for Increased Thermostability Fraser, Nicholas J.; Liu, Jian-Wei; Mabbitt, Peter D. Journal of Molecular Biology, Vol. 428, Issue 11 https://doi.org/10.1016/j.jmb.2016.03.014	journal	June 2016
How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes Rauwerdink, Alissa; Kazlauskas, Romas J. ACS Catalysis, Vol. 5, Issue 10 https://doi.org/10.1021/acscatal.5b01539	journal	September 2015
Alpha/beta-hydrolases: A unique structural motif coordinates catalytic acid residue in 40 protein fold families Dimitriou, Polytimi S.; Denesyuk, Alexander; Takahashi, Seiji Proteins: Structure, Function, and Bioinformatics, Vol. 85, Issue 10 https://doi.org/10.1002/prot.25338	journal	July 2017
α/β Hydrolase fold enzymes: the family keeps growing Nardini, Marco; Dijkstra, Bauke W. Current Opinion in Structural Biology, Vol. 9, Issue 6 https://doi.org/10.1016/S0959-440X(99)00037-8	journal	December 1999
Oligomeric interactions maintain active‐site structure in a noncooperative enzyme family Li, Yaohui; Zhang, Rongzhen; Wang, Chi The EMBO Journal, Vol. 41, Issue 17 https://doi.org/10.15252/embj.2021108368	journal	July 2022
Assembly reflects evolution of protein complexes Levy, Emmanuel D.; Erba, Elisabetta Boeri; Robinson, Carol V. Nature, Vol. 453, Issue 7199 https://doi.org/10.1038/nature06942	journal	June 2008
Contribution of Electrostatic Interactions, Compactness and Quaternary Structure to Protein Thermostability: Lessons from Structural Genomics of Thermotoga maritima Robinson-Rechavi, Marc; Alibés, Andreu; Godzik, Adam Journal of Molecular Biology, Vol. 356, Issue 2 https://doi.org/10.1016/j.jmb.2005.11.065	journal	February 2006
Modulation of protein oligomerization: An overview Kumari, Neha; Yadav, Savita Progress in Biophysics and Molecular Biology, Vol. 149 https://doi.org/10.1016/j.pbiomolbio.2019.03.003	journal	December 2019
Disulfide Bond in Pseudomonas aeruginosa Lipase Stabilizes the Structure but Is Not Required for Interaction with Its Foldase Liebeton, Klaus; Zacharias, Annette; Jaeger, Karl-Erich Journal of Bacteriology, Vol. 183, Issue 2 https://doi.org/10.1128/JB.183.2.597-603.2001	journal	January 2001
Structural insight into catalytic mechanism of PET hydrolase Han, Xu; Liu, Weidong; Huang, Jian-Wen Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/s41467-017-02255-z	journal	December 2017
An engineered PET depolymerase to break down and recycle plastic bottles Tournier, V.; Topham, C. M.; Gilles, A. Nature, Vol. 580, Issue 7802 https://doi.org/10.1038/s41586-020-2149-4	journal	April 2020
Optimization of the Turnover in Artificial Enzymes via Directed Evolution Results in the Coupling of Protein Dynamics to Chemistry Schafer, Joseph W.; Zoi, Ioanna; Antoniou, Dimitri Journal of the American Chemical Society, Vol. 141, Issue 26 https://doi.org/10.1021/jacs.9b04515	journal	June 2019
Protein Dynamics and Enzymatic Catalysis Schwartz, Steven D. The Journal of Physical Chemistry B, Vol. 127, Issue 12 https://doi.org/10.1021/acs.jpcb.3c00477	journal	March 2023
Glutathione redox potential is low and glutathionylated and cysteinylated hemoglobin levels are elevated in maintenance hemodialysis patients Khazim, Khaled; Giustarini, Daniela; Rossi, Ranieri Translational Research, Vol. 162, Issue 1 https://doi.org/10.1016/j.trsl.2012.12.014	journal	July 2013
An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments Aitken, Colin Echeverría; Marshall, R. Andrew; Puglisi, Joseph D. Biophysical Journal, Vol. 94, Issue 5 https://doi.org/10.1529/biophysj.107.117689	journal	March 2008
Selective reduction of disulfides by tris(2-carboxyethyl)phosphine Burns, John A.; Butler, James C.; Moran, John The Journal of Organic Chemistry, Vol. 56, Issue 8 https://doi.org/10.1021/jo00008a014	journal	April 1991
Broad Analysis of Vicinal Disulfides: Occurrences, Conformations with Cis or with Trans Peptides, and Functional Roles Including Sugar Binding Richardson, Jane S.; Videau, Lizbeth L.; Williams, Christopher J. Journal of Molecular Biology, Vol. 429, Issue 9 https://doi.org/10.1016/j.jmb.2017.03.017	journal	May 2017
The Redox-sensing Transcriptional Regulator RexT Controls Expression of Thioredoxin A2 in the Cyanobacterium Anabaena sp. Strain PCC 7120 Ehira, Shigeki; Ohmori, Masayuki Journal of Biological Chemistry, Vol. 287, Issue 48 https://doi.org/10.1074/jbc.M112.384206	journal	November 2012
Thiol-based redox control of enzymes involved in the tetrapyrrole biosynthesis pathway in plants Richter, Andreas S.; Grimm, Bernhard Frontiers in Plant Science, Vol. 4 https://doi.org/10.3389/fpls.2013.00371	journal	January 2013
The C-terminal cysteine-rich motif of NYE1/SGR1 is indispensable for its function in chlorophyll degradation in Arabidopsis Xie, Zuokun; Wu, Shengdong; Chen, Junyi Plant Molecular Biology, Vol. 101, Issue 3 https://doi.org/10.1007/s11103-019-00902-1	journal	July 2019
Modification of cysteine residues in the ChlI and ChlH subunits of magnesium chelatase results in enzyme inactivation Jensen, Poul E.; Reid, James D.; Hunter, C. Neil Biochemical Journal, Vol. 352, Issue 2 https://doi.org/10.1042/bj3520435	journal	November 2000
The CHLI1 Subunit of Arabidopsis thaliana Magnesium Chelatase Is a Target Protein of the Chloroplast Thioredoxin Ikegami, Akinori; Yoshimura, Naho; Motohashi, Ken Journal of Biological Chemistry, Vol. 282, Issue 27 https://doi.org/10.1074/jbc.M703324200	journal	July 2007
Arabidopsis Mg-Protoporphyrin IX Methyltransferase Activity and Redox Regulation Depend on Conserved Cysteines Richter, Andreas S.; Wang, Peng; Grimm, Bernhard Plant and Cell Physiology, Vol. 57, Issue 3 https://doi.org/10.1093/pcp/pcw007	journal	January 2016
Detecting sequence signals in targeting peptides using deep learning Almagro Armenteros, Jose Juan; Salvatore, Marco; Emanuelsson, Olof Life Science Alliance, Vol. 2, Issue 5 https://doi.org/10.26508/lsa.201900429	journal	September 2019
Rieske Oxygenase Catalyzed C–H Bond Functionalization Reactions in Chlorophyll b Biosynthesis Liu, Jianxin; Knapp, Madison; Jo, Minshik ACS Central Science, Vol. 8, Issue 10 https://doi.org/10.1021/acscentsci.2c00058	journal	July 2022
Purification and Properties of Chlorophyllase from Greened Rye Seedlings1 Tanaka, Koichiro; Kakuno, Tomisaburo; Yamashita, Jinpei The Journal of Biochemistry, Vol. 92, Issue 6 https://doi.org/10.1093/oxfordjournals.jbchem.a134106	journal	October 1982
Purification and Properties of Chlorophyllase from Ailanthus altissima (Tree-of-Heaven) McFeeters, Roger F.; Chichester, Clinton O.; Whitaker, John R. Plant Physiology, Vol. 47, Issue 5 https://doi.org/10.1104/pp.47.5.609	journal	May 1971
Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks Shannon, P. Genome Research, Vol. 13, Issue 11 https://doi.org/10.1101/gr.1239303	journal	November 2003

Similar Records

A structure-function analysis of chlorophyllase reveals a mechanism for activity regulation dependent on disulfide bonds

Journal Article · Tue Feb 28 00:00:00 EST 2023 · Journal of Biological Chemistry · OSTI ID:1923104

Jo, Minshik; Knapp, Madison; Boggs, David G.; +3 more

Related Subjects

chlorophyll
photosynthesis
photosynthetic pigment
lipase
enzyme catalysis
hydrolase
enzyme kinetics
disulfides
dyes and pigments
hydrolysis
monomers
peptides and proteins

Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis

Citation Formats

References (65)

Similar Records

Related Subjects