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

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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
https://doi.org/10.1021/bi035734d
Copyright © 2004 American Chemical Society

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    Abstract

    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.

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     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.

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    Calculated thermodynamic constants and electronic energy of 8-substituted FAD analogues. This material is available free of charge via the Internet at http://pubs.acs.org.

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