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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.


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