Date

May 2011

Document Type

Thesis

Degree Name

M.S.

Institution

Oregon Health & Science University

Abstract

Manganese (III, IV) oxides are strong naturally occurring oxidants that possess the ability to control the distribution of many trace metals in the environment. Sequence homology of Mn(II) oxidase enzymes with multicopper oxidases and genetic manipulation suggest that two different multi-copper oxidases are the dominant enzymes capable of Mn(II) oxidation in Pseudomonas putida GB-1. However, the functional role and enzymatic mechanism remains unknown. Complete cellular fractionation of Pseudomonas putida GB-1 localized Mn(II) oxidizing activity to the outer membrane. Native PAGE demonstrated that the Mn(II) oxidase activity has an apparent molecular weight of >250kDa. Fast protein liquid chromatography using hydrophobic interaction and size exclusion resulted in a partially purified fraction of 130kDa. Tandem MS/MS of the partially pure fraction identified a type two multi-copper oxidase (Mco). However, when mco is deleted, Mn(II) oxidation still occurs, consistent with the notion that more than one enzyme is responsible for Mn(II) oxidation. In vitro experiments with active membrane fractions demonstrate that reactive oxygen species have a role in Mn(II) oxidation. In particular, superoxide dismutase and copper greatly inhibit Mn(II) oxidation, suggesting that superoxide radicals are involved. Hydrogen peroxide also appears to be part of the enzymatic mechanism based on the ability of catalase to inhibit Mn(II) oxidation. These results suggest that the enzymatic oxidation of manganese requires superoxide radical formation and the utilization of hydrogen peroxide for proper catalysis. Further analysis of MS/MS data identified hypothetical proteins 2552 and 2553 in the partially purified active fraction. Over expression of these hypothetical proteins and the in-frame deletion of 2552 demonstrate that these proteins have an essential role in Mn(II) oxidation in P.putida GB-1. Transformation studies suggest an association of the multi-copper oxidase, MnxG with both hypothetical proteins. The fact that 2553 is transcriptionally regulated by the response regulator MnxR provides further evidence that these hypothetical proteins are important for Mn (II) oxidation. However, the exact role and function of these proteins remain unknown. This is the first indication that other outer membrane proteins besides the Mn(II) oxidase(s) are involved with Mn(II) oxidation in Pseudomonas putida GB-1.

Identifier

doi:10.6083/M41Z42C2

Division

Institute of Environmental Health

School

School of Medicine

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