This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Xun, L.
Right arrow Articles by Sandvik, E. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Xun, L.
Right arrow Articles by Sandvik, E. R.
Agricola
Right arrow Articles by Xun, L.
Right arrow Articles by Sandvik, E. R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, February 2000, p. 481-486, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

Luying Xun* and Erik R. Sandvik

School of Molecular Biosciences, Washington State University, Pullman, Washington 99164-4234

Received 19 August 1999/Accepted 9 November 1999

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH2)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH2 in vitro, HpaB was able to use FADH2 and O2 for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH2 for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH2-utilizing monooxygenase. FADH2 generated by Fre was rapidly oxidized by O2 to form H2O2 in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH2 and transitorily protected it from rapid autoxidation by O2. When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O2 for FADH2 utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH2 produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH2 was autoxidized by O2, causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH2-utilizing monooxygenase that uses FADH2 as a substrate rather than as a cofactor.

* Corresponding author. Mailing address: School of Molecular Biosciences, Washington State University, Pullman, WA 99163-4234. Phone: (509) 335-2787. Fax: (509) 335-1907. E-mail: xun{at}mail.wsu.edu.

Applied and Environmental Microbiology, February 2000, p. 481-486, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Takeo, M., Murakami, M., Niihara, S., Yamamoto, K., Nishimura, M., Kato, D.-i., Negoro, S. (2008). Mechanism of 4-Nitrophenol Oxidation in Rhodococcus sp. Strain PN1: Characterization of the Two-Component 4-Nitrophenol Hydroxylase and Regulation of Its Expression. J. Bacteriol. 190: 7367-7374 [Abstract] [Full Text]  
  • Nissen, M. S., Youn, B., Knowles, B. D., Ballinger, J. W., Jun, S.-Y., Belchik, S. M., Xun, L., Kang, C. (2008). Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis. J. Biol. Chem. 283: 28710-28720 [Abstract] [Full Text]  
  • Clarke-Pearson, M. F., Brady, S. F. (2008). Paerucumarin, a New Metabolite Produced by the pvc Gene Cluster from Pseudomonas aeruginosa. J. Bacteriol. 190: 6927-6930 [Abstract] [Full Text]  
  • Valton, J., Mathevon, C., Fontecave, M., Niviere, V., Ballou, D. P. (2008). Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolor. J. Biol. Chem. 283: 10287-10296 [Abstract] [Full Text]  
  • Belchik, S. M., Xun, L. (2008). Functions of Flavin Reductase and Quinone Reductase in 2,4,6-Trichlorophenol Degradation by Cupriavidus necator JMP134. J. Bacteriol. 190: 1615-1619 [Abstract] [Full Text]  
  • Lee, J.-K., Zhao, H. (2007). Identification and Characterization of the Flavin:NADH Reductase (PrnF) Involved in a Novel Two-Component Arylamine Oxygenase. J. Bacteriol. 189: 8556-8563 [Abstract] [Full Text]  
  • Kim, S.-H., Hisano, T., Takeda, K., Iwasaki, W., Ebihara, A., Miki, K. (2007). Crystal Structure of the Oxygenase Component (HpaB) of the 4-Hydroxyphenylacetate 3-Monooxygenase from Thermus thermophilus HB8. J. Biol. Chem. 282: 33107-33117 [Abstract] [Full Text]  
  • Perry, L. L., Zylstra, G. J. (2007). Cloning of a Gene Cluster Involved in the Catabolism of p-Nitrophenol by Arthrobacter sp. Strain JS443 and Characterization of the p-Nitrophenol Monooxygenase. J. Bacteriol. 189: 7563-7572 [Abstract] [Full Text]  
  • Vallurupalli, P., Kay, L. E. (2006). Complementarity of ensemble and single-molecule measures of protein motion: A relaxation dispersion NMR study of an enzyme complex. Proc. Natl. Acad. Sci. USA 103: 11910-11915 [Abstract] [Full Text]  
  • Sucharitakul, J., Chaiyen, P., Entsch, B., Ballou, D. P. (2006). Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii. J. Biol. Chem. 281: 17044-17053 [Abstract] [Full Text]  
  • Weir, K. M., Sutherland, T. D., Horne, I., Russell, R. J., Oakeshott, J. G. (2006). A Single Monooxygenase, Ese, Is Involved in the Metabolism of the Organochlorides Endosulfan and Endosulfate in an Arthrobacter sp.. Appl. Environ. Microbiol. 72: 3524-3530 [Abstract] [Full Text]  
  • Valton, J., Fontecave, M., Douki, T., Kendrew, S. G., Niviere, V. (2006). An Aromatic Hydroxylation Reaction Catalyzed by a Two-component FMN-dependent Monooxygenase: THE ActVA-ActVB SYSTEM FROM STREPTOMYCES COELICOLOR. J. Biol. Chem. 281: 27-35 [Abstract] [Full Text]  
  • Arias-Barrau, E., Sandoval, A., Naharro, G., Olivera, E. R., Luengo, J. M. (2005). A Two-component Hydroxylase Involved in the Assimilation of 3-Hydroxyphenyl Acetate in Pseudomonas putida. J. Biol. Chem. 280: 26435-26447 [Abstract] [Full Text]  
  • Valton, J., Filisetti, L., Fontecave, M., Niviere, V. (2004). A Two-component Flavin-dependent Monooxygenase Involved in Actinorhodin Biosynthesis in Streptomyces coelicolor. J. Biol. Chem. 279: 44362-44369 [Abstract] [Full Text]  
  • Otto, K., Hofstetter, K., Rothlisberger, M., Witholt, B., Schmid, A. (2004). Biochemical Characterization of StyAB from Pseudomonas sp. Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase. J. Bacteriol. 186: 5292-5302 [Abstract] [Full Text]  
  • Xun, L., Webster, C. M. (2004). A Monooxygenase Catalyzes Sequential Dechlorinations of 2,4,6-Trichlorophenol by Oxidative and Hydrolytic Reactions. J. Biol. Chem. 279: 6696-6700 [Abstract] [Full Text]  
  • Kirchner, U., Westphal, A. H., Muller, R., van Berkel, W. J. H. (2003). Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD. J. Biol. Chem. 278: 47545-47553 [Abstract] [Full Text]  
  • Gisi, M. R., Xun, L. (2003). Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:Flavin Adenine Dinucleotide Oxidoreductase (TftC) of Burkholderia cepacia AC1100. J. Bacteriol. 185: 2786-2792 [Abstract] [Full Text]  
  • Louie, T. M., Yang, H., Karnchanaphanurach, P., Xie, X. S., Xun, L. (2002). FAD Is a Preferred Substrate and an Inhibitor of Escherichia coli General NAD(P)H:Flavin Oxidoreductase. J. Biol. Chem. 277: 39450-39455 [Abstract] [Full Text]  
  • Woodmansee, A. N., Imlay, J. A. (2002). Reduced Flavins Promote Oxidative DNA Damage in Non-respiring Escherichia coli by Delivering Electrons to Intracellular Free Iron. J. Biol. Chem. 277: 34055-34066 [Abstract] [Full Text]  
  • Louie, T. M., Webster, C. M., Xun, L. (2002). Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134. J. Bacteriol. 184: 3492-3500 [Abstract] [Full Text]  
  • Diaz, E., Ferrandez, A., Prieto, M. A., Garcia, J. L. (2001). Biodegradation of Aromatic Compounds by Escherichia coli. Microbiol. Mol. Biol. Rev. 65: 523-569 [Abstract] [Full Text]  
  • Bohuslavek, J., Payne, J. W., Liu, Y., Bolton, H. Jr., Xun, L. (2001). Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1. Appl. Environ. Microbiol. 67: 688-695 [Abstract] [Full Text]  
  • Liu, Y., Louie, T. M., Payne, J., Bohuslavek, J., Bolton, H. Jr., Xun, L. (2001). Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1. Appl. Environ. Microbiol. 67: 696-701 [Abstract] [Full Text]  
  • Delaney, S. M., Mavrodi, D. V., Bonsall, R. F., Thomashow, L. S. (2001). phzO, a Gene for Biosynthesis of 2-Hydroxylated Phenazine Compounds in Pseudomonas aureofaciens 30-84. J. Bacteriol. 183: 318-327 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»