ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Use of 8-Substituted-FAD Analogues To Investigate the Hydroxylation Mechanism of the Flavoprotein 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase,

View Author Information
Department of Biochemistry and Center for Protein Structure & Function, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok, Thailand, and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan
Cite this: Biochemistry 2004, 43, 13, 3933–3943
Publication Date (Web):March 2, 2004
Copyright © 2004 American Chemical Society

    Article Views





    Other access options
    Supporting Info (1)»


    2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) is a flavoprotein that catalyzes the oxygenation of MHPC to form α-(N-acetylaminomethylene)-succinic acid. Although formally similar to the oxygenation reactions catalyzed by phenol hydroxylases, MHPCO catalyzes the oxygenation of a pyridyl derivative rather than a simple phenol. Therefore, in this study, the mechanism of the reaction was investigated by replacing the natural cofactor FAD with FAD analogues having various substituents (−Cl, −CN, −NH2, −OCH3) at the C8-position of the isoalloxazine. Thermodynamic and catalytic properties of the reconstituted enzyme were investigated and found to be similar to those of the native enzyme, validating that these FAD analogues are reasonable to be used as mechanistic probes. Dissociation constants for the binding of MHPC or the substrate analogue 5-hydroxynicotinate (5HN) to the reconstituted enzymes indicate that the reconstituted enzymes bind well with ligands. Redox potential values of the reconstituted enzymes were measured and found to be more positive than the values of free FAD analogues, which correlated well with the electronic effects of the 8-substituents. Studies of the reductive half-reaction of MHPCO have shown that the rates of flavin reduction by NADH could be described as a parabolic relationship with the redox potential values of the reconstituted enzymes, which is consistent with the Marcus electron transfer theory. Studies of the oxidative half-reaction of MHPCO revealed that the rate of hydroxylation depended upon the different analogues employed. The rate constants for the hydroxylation step correlated with the calculated pKa values of the 8-substituted C(4a)-hydroxyflavin intermediates, which are the leaving groups in the oxygen transfer step. It was observed that the rates of hydroxylation were greater when the pKa values of C(4a)-hydroxyflavins were lower. Although these results are not as dramatic as those from analogous studies with parahydroxybenzoate hydroxylase (Ortiz-Maldonado et al., (1999) Biochemistry38, 8124−8137), they are consistent with the model that the oxygenation reaction of MHPCO occurs via an electrophilic aromatic substitution mechanism analogous to the mechanisms for parahydroxybenzoate and phenol hydroxylases.

    Read this article

    To access this article, please review the available access options below.

    Get instant access

    Purchase Access

    Read this article for 48 hours. Check out below using your ACS ID or as a guest.


    Access through Your Institution

    You may have access to this article through your institution.

    Your institution does not have access to this content. You can change your affiliated institution below.

     Financial support was received from Thailand Research Fund Grants RSA/09/2545 and RTA/02/2544 and Mahidol University (to P.C.) and NIH Grant GM64711 (to D.P.B.).

     This paper is dedicated to the memory of our friend, colleague, and mentor, Vincent Massey, deceased Aug 26, 2002.


     To whom correspondence should be addressed. Phone:  662-201-5607. Fax:  662-248-0375. E-mail:  [email protected].

     Mahidol University.


      University of Michigan.

    Supporting Information Available

    Jump To

    Calculated thermodynamic constants and electronic energy of 8-substituted FAD analogues. This material is available free of charge via the Internet at

    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system:

    Cited By

    This article is cited by 38 publications.

    1. Amrita B. Hazra, David P. Ballou, Michiko E. Taga. Unique Biochemical and Sequence Features Enable BluB To Destroy Flavin and Distinguish BluB from the Flavin Monooxygenase Superfamily. Biochemistry 2018, 57 (11) , 1748-1757.
    2. Syam Sundar Neti and C. Dale Poulter . Site-Selective Synthesis of 15N- and 13C-Enriched Flavin Mononucleotide Coenzyme Isotopologues. The Journal of Organic Chemistry 2016, 81 (12) , 5087-5092.
    3. Pirom Chenprakhon, Bhinyo Panijpan, and Pimchai Chaiyen . An Experiment Illustrating the Change in Ligand pKa upon Protein Binding. Journal of Chemical Education 2012, 89 (6) , 791-795.
    4. Boxue Tian, Åke Strid, and Leif A. Eriksson . Catalytic Roles of Active-Site Residues in 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase: An ONIOM/DFT Study. The Journal of Physical Chemistry B 2011, 115 (8) , 1918-1926.
    5. Kathryn M. McCulloch, Tathagata Mukherjee, Tadhg P. Begley and Steven E. Ealick. Structure of the PLP Degradative Enzyme 2-Methyl-3-hydroxypyridine-5-carboxylic Acid Oxygenase from Mesorhizobium loti MAFF303099 and Its Mechanistic Implications. Biochemistry 2009, 48 (19) , 4139-4149.
    6. Jeerus Sucharitakul, Methinee Prongjit, Dietmar Haltrich and Pimchai Chaiyen . Detection of a C4a-Hydroperoxyflavin Intermediate in the Reaction of a Flavoprotein Oxidase. Biochemistry 2008, 47 (33) , 8485-8490.
    7. Tathagata Mukherjee, David G. Hilmey and Tadhg P. Begley. PLP Catabolism: Identification of the 2-(Acetamidomethylene)succinate Hydrolase Gene in Mesorhizobium loti MAFF303099. Biochemistry 2008, 47 (23) , 6233-6241.
    8. Fangjie Guo, Yilin Tian, Shujing Ji, Hao Min, Wen Ding, Haiying Yu, Yingqi Li, Li Ji. Environmental biotransformation mechanisms by flavin-dependent monooxygenase: A computational study. Chemosphere 2023, 325 , 138403.
    9. Fangjie Guo, Yilin Tian, Shujing Ji, Hao Min, Wen Ding, Haiying Yu, Yingqi Li, Li Ji. Environmental Biotransformation Mechanisms by Flavin-Dependent Monooxygenase: A Computational Study. SSRN Electronic Journal 2022, 336
    10. Patricia Ferreira, Milagros Medina. Anaerobic Stopped-Flow Spectrophotometry with Photodiode Array Detection in the Presteady State: An Application to Elucidate Oxidoreduction Mechanisms in Flavoproteins. 2021, 135-155.
    11. Warintra Pitsawong, Pirom Chenprakhon, Taweesak Dhammaraj, Dheeradhach Medhanavyn, Jeerus Sucharitakul, Chanakan Tongsook, Willem J.H. van Berkel, Pimchai Chaiyen, Anne-Frances Miller. Tuning of pK values activates substrates in flavin-dependent aromatic hydroxylases. Journal of Biological Chemistry 2020, 295 (12) , 3965-3981.
    12. Pirom Chenprakhon, Panu Pimviriyakul, Chanakan Tongsook, Pimchai Chaiyen. Phenolic hydroxylases. 2020, 283-326.
    13. Ruchanok Tinikul, Paweenapon Chunthaboon, Jittima Phonbuppha, Tanakan Paladkong. Bacterial luciferase: Molecular mechanisms and applications. 2020, 427-455.
    14. Panu Pimviriyakul, Pimchai Chaiyen. Overview of flavin-dependent enzymes. 2020, 1-36.
    15. Pirom Chenprakhon, Thanyaporn Wongnate, Pimchai Chaiyen. Monooxygenation of aromatic compounds by flavin‐dependent monooxygenases. Protein Science 2019, 28 (1) , 8-29.
    16. Panu Pimviriyakul, Panida Surawatanawong, Pimchai Chaiyen. Oxidative dehalogenation and denitration by a flavin-dependent monooxygenase is controlled by substrate deprotonation. Chemical Science 2018, 9 (38) , 7468-7482.
    17. Tomoya Hino, Haruka Hamamoto, Hirokazu Suzuki, Hisashi Yagi, Takashi Ohshiro, Shingo Nagano. Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity. Journal of Biological Chemistry 2017, 292 (38) , 15804-15813.
    18. Pirom Chenprakhon, Taweesak Dhammaraj, Rattikan Chantiwas, Pimchai Chaiyen. Hydroxylation of 4-hydroxyphenylethylamine derivatives by R263 variants of the oxygenase component of p -hydroxyphenylacetate-3-hydroxylase. Archives of Biochemistry and Biophysics 2017, 620 , 1-11.
    19. Thikumporn Luanloet, Jeerus Sucharitakul, Pimchai Chaiyen. Selectivity of substrate binding and ionization of 2‐methyl‐3‐hydroxypyridine‐5‐carboxylic acid oxygenase. The FEBS Journal 2015, 282 (16) , 3107-3125.
    20. Tatiana V. Mishanina, Amnon Kohen. Synthesis and application of isotopically labeled flavin nucleotides. Journal of Labelled Compounds and Radiopharmaceuticals 2015, 58 (9) , 370-375.
    21. Luuk J.G.W. van Wilderen, Gary Silkstone, Maria Mason, Jasper J. van Thor, Michael T. Wilson. Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen. FEBS Open Bio 2015, 5 (1) , 885-892.
    22. Jesús I. Martínez, Pablo J. Alonso, Inés García-Rubio, Milagros Medina. Methyl rotors in flavoproteins. Phys. Chem. Chem. Phys. 2014, 16 (47) , 26203-26212.
    23. Jeerus Sucharitakul, Chanakan Tongsook, Danaya Pakotiprapha, Willem J.H. van Berkel, Pimchai Chaiyen. The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle*. Journal of Biological Chemistry 2013, 288 (49) , 35210-35221.
    24. Chanakan Tongsook, Jeerus Sucharitakul, Kittisak Thotsaporn, Pimchai Chaiyen. Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase. Journal of Biological Chemistry 2011, 286 (52) , 44491-44502.
    25. Tathagata Mukherjee, Jeremiah Hanes, Ivo Tews, Steven E. Ealick, Tadhg P. Begley. Pyridoxal phosphate: Biosynthesis and catabolism. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2011, 1814 (11) , 1585-1596.
    26. Stefania Montersino, Dirk Tischler, George T. Gassner, Willem J. H. van Berkel. Catalytic and Structural Features of Flavoprotein Hydroxylases and Epoxidases. Advanced Synthesis & Catalysis 2011, 353 (13) , 2301-2319.
    27. Niall A. McDonald, Chandramouleeswaran Subramani, Stuart T. Caldwell, Nada Y. Zainalabdeen, Graeme Cooke, Vincent M. Rotello. Simultaneous hydrogen bonding and π-stacking interactions between flavin/porphyrin host–guest systems. Tetrahedron Letters 2011, 52 (17) , 2107-2110.
    28. Nantidaporn Ruangchan, Chanakan Tongsook, Jeerus Sucharitakul, Pimchai Chaiyen. pH-dependent Studies Reveal an Efficient Hydroxylation Mechanism of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase. Journal of Biological Chemistry 2011, 286 (1) , 223-233.
    29. Boxue Tian, Yaoquan Tu, Åke Strid, Leif A. Eriksson. Hydroxylation and Ring‐Opening Mechanism of an Unusual Flavoprotein Monooxygenase, 2‐Methyl‐3‐hydroxypyridine‐5‐carboxylic Acid Oxygenase: A Theoretical Study. Chemistry – A European Journal 2010, 16 (8) , 2557-2566.
    30. Tathagata Mukherjee, Kathryn M. McCulloch, Steven E. Ealick, Tadhg P. Begley. Cofactor Catabolism. 2010, 649-674.
    31. Pimchai Chaiyen. Flavoenzymes catalyzing oxidative aromatic ring-cleavage reactions. Archives of Biochemistry and Biophysics 2010, 493 (1) , 62-70.
    32. Andrea Alfieri, Francesco Fersini, Nantidaporn Ruangchan, Methinee Prongjit, Pimchai Chaiyen, Andrea Mattevi. Structure of the monooxygenase component of a two-component flavoprotein monooxygenase. Proceedings of the National Academy of Sciences 2007, 104 (4) , 1177-1182.
    33. Baiqiang Yuan, Nana Yokochi, Yu Yoshikane, Kouhei Ohnishi, Toshiharu Yagi. Molecular cloning, identification and characterization of 2-methyl-3-hydroxypyridine-5-carboxylic-acid-dioxygenase-coding gene from the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. Journal of Bioscience and Bioengineering 2006, 102 (6) , 504-510.
    34. Jeerus Sucharitakul, Pimchai Chaiyen, Barrie Entsch, David P. Ballou. Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii. Journal of Biological Chemistry 2006, 281 (25) , 17044-17053.
    35. Greanggrai Hommalai, Pimchai Chaiyen, Jisnuson Svasti. Studies on the transglucosylation reactions of cassava and Thai rosewood β-glucosidases using 2-deoxy-2-fluoro-glycosyl-enzyme intermediates. Archives of Biochemistry and Biophysics 2005, 442 (1) , 11-20.
    36. Joseph B. Carroll, Brian J. Jordan, Hao Xu, Belma Erdogan, Lisa Lee, Lily Cheng, Christopher Tiernan, Graeme Cooke, Vincent M. Rotello. Model Systems for Flavoenzyme Activity:  Site-Isolated Redox Behavior in Flavin-Functionalized Random Polystyrene Copolymers. Organic Letters 2005, 7 (13) , 2551-2554.
    37. Barrie Entsch, Lindsay J. Cole, David P. Ballou. Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase. Archives of Biochemistry and Biophysics 2005, 433 (1) , 297-311.
    38. Igor Efimov, William S. McIntire. Relationship between Charge-Transfer Interactions, Redox Potentials, and Catalysis for Different Forms of the Flavoprotein Component of p- Cresol Methylhydroxylase. Journal of the American Chemical Society 2005, 127 (2) , 732-741.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    You’ve supercharged your research process with ACS and Mendeley!

    STEP 1:
    Click to create an ACS ID

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Your Mendeley pairing has expired. Please reconnect