Studies on the Mechanism of p-Hydroxyphenylacetate 3-Hydroxylase from Pseudomonas aeruginosa: A System Composed of a Small Flavin Reductase and a Large Flavin-Dependent Oxygenase
- 10 December 2009
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 49 (2), 372-385
- https://doi.org/10.1021/bi901454u
Abstract
There are two known types of microbial two-component flavin-dependent monooxygenases that catalyze oxygenation of p-hydroxyphenylacetate (HPA), and they are distinguished by having structurally distinct reductases and oxygenases. This paper presents a detailed analysis of the properties of the enzyme from Pseudomonas aeruginosa, an example of one group, and compares its properties to those published for the Acinetobacter baumannii enzyme, an example of the alternative group. The reductase and oxygenase from P. aeruginosa were expressed in Escherichia coli. The reductase was purified as a stable C-terminally His-tagged yellow protein containing weakly bound FAD, and the oxygenase was purified as a stable colorless N-terminally His-tagged protein. The reductase catalyzes the reduction of FAD by NADH and releases the FADH− product into solution, but unlike the reductase from A. baumannii, this catalysis is not influenced by HPA. The oxygenase binds the released FADH− and catalyzes the oxygenation of HPA to form 3,4-dihydroxyphenylacetate, after which the FAD dissociates to be re-reduced by the reductase, a common overall pattern for two-component flavin-dependent oxygenases. With this system, it appears that interactions between the reductase and the oxygenase can facillitate the transfer of FADH− to the oxygenase, although they are not required. We show that the P. aeruginosa oxygenase system in complex with FADH− reacts with O2 to form a quasi-stable, unusually high-extinction flavin hydroperoxide species that binds HPA and reacts to form the product. The resultant flavin hydroxide decomposes to FAD and water while still bound to the oxygenase and then releases product and FAD from the protein. Unlike the enzyme from A. baumannii, during normal catalysis involving both the reductase and oxygenase, the rate-determining step in catalysis is the dissociation of FAD from the oxygenase in a process that is independent of the concentration of HPA. Structures for the reductases and oxygenases from A. baumannii and from Thermus thermophilus (similar to the P. aeruginosa system) form a basis for interpreting the molecular origins of the differences between the two groups of flavin-dependent two-component oxygenases.Keywords
This publication has 25 references indexed in Scilit:
- Fre Is the Major Flavin Reductase Supporting Bioluminescence from Vibrio harveyi Luciferase in Escherichia coliPublished by Elsevier BV ,2009
- Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenaseProceedings of the National Academy of Sciences of the United States of America, 2008
- Mechanism and Regulation of the Two-component FMN-dependent Monooxygenase ActVA-ActVB from Streptomyces coelicolorPublished by Elsevier BV ,2008
- Kinetics of a Two-Component p-Hydroxyphenylacetate Hydroxylase Explain How Reduced Flavin Is Transferred from the Reductase to the OxygenaseBiochemistry, 2007
- Structure of the monooxygenase component of a two-component flavoprotein monooxygenaseProceedings of the National Academy of Sciences of the United States of America, 2007
- Flavoprotein monooxygenases, a diverse class of oxidative biocatalystsJournal of Biotechnology, 2006
- Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumanniiPublished by Elsevier BV ,2006
- Coordinated Production and Utilization of FADH2 by NAD(P)H−Flavin Oxidoreductase and 4-Hydroxyphenylacetate 3-MonooxygenaseBiochemistry, 2003
- Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 Å resolution: Analysis of the enzyme-substrate and enzyme-product complexesJournal of Molecular Biology, 1989
- Oxidation of reduced flavins by quinonesBiochemical and Biophysical Research Communications, 1969