Proteomic analysis of mouse kidney peroxisomes: identification of RP2p as a peroxisomal nudix hydrolase with acyl-CoA diphosphatase activity

*Department of Clinical Chemistry, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE, Amsterdam, The Netherlands

†Department of Medical Biochemistry, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE, Amsterdam, The Netherlands

‡Department of Pediatrics, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE, Amsterdam, The Netherlands

^{To whom correspondence should be addressed (email}ln.avu.cma@srednaW.J.R).

Received 2005 Jun 2; Revised 2005 Sep 20; Accepted 2005 Sep 27.

Abstract

Proteomic analysis of mouse kidney peroxisomes resulted in the identification of a novel nudix hydrolase designated RP2p, which is encoded by the D7RP2e gene. RP2p consists of 357 amino acids and contains two conserved domains: a nudix hydrolase domain and a CoA-binding domain. In addition, a PTS (peroxisomal targeting signal) type 1 (Ala-His-Leu) was found at the C-terminus. Analysis of the enzyme characteristics revealed that RP2p is a CoA diphosphatase with activity towards CoA, oxidized CoA and a wide range of CoA esters, including choloyl-CoA and branched-chain fatty-acyl-CoA esters. The enzymatic properties of RP2p indicate that at low substrate concentrations medium and long-chain fatty-acyl-CoA esters are the primary substrates. Enzyme activity was optimal at pH 9 or above, and required the presence of Mg^{or Mn}^{ions. Subcellular fractionation studies revealed that all CoA diphosphatase activity in mouse kidney is restricted to peroxisomes.}

Keywords: acyl-CoA ester, kidney, mouse, nudix hydrolase, peroxisome, proteomics

Abbreviations: BN, Blue native; DTT, dithiothreitol; IPTG, isopropyl β-D-thiogalactoside; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; MBP, maltose-binding protein; PTS, peroxisomal targeting signal; Q-TOF, quadrupole time-of-flight

Abstract

Acknowledgments

We gratefully acknowledge Dr Hans Waterham and Dr Stephan Kemp for critically reading the manuscript. We thank Lida Zoetekouw and Simone Denis for expert technical assistance and H. L. Dekker for expert Q-TOF MS/MS analysis. This work was supported by the FP6 European Union Project ‘Peroxisomes’ (LSHG-CT-2004-512018).

Acknowledgments

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