HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 19, 14316-14327, May 11, 2007

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 282/19/14316    most recent M700912200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by White, T. A. Articles by Tanner, J. J. Search for Related Content PubMed PubMed Citation Articles by White, T. A. Articles by Tanner, J. J. Social Bookmarking                      What's this?

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus^*

Tommi A. White, Navasona Krishnan, Donald F. Becker, and John J. Tanner^¶1

From the Departments of Biochemistry and ^¶Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211 and the Department of Biochemistry, Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588

Proline dehydrogenase (PRODH) and ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) catalyze the two-step oxidation of proline to glutamate. They are distinct monofunctional enzymes in all eukaryotes and some bacteria but are fused into bifunctional enzymes known as proline utilization A (PutA) in other bacteria. Here we report the first structure and biochemical data for a monofunctional PRODH. The 2.0-Å resolution structure of Thermus thermophilus PRODH reveals a distorted ()₈ barrel catalytic core domain and a hydrophobic -helical domain located above the carboxyl-terminal ends of the strands of the barrel. Although the catalytic core is similar to that of the PutA PRODH domain, the FAD conformation of T. thermophilus PRODH is remarkably different and likely reflects unique requirements for membrane association and communication with P5CDH. Also, the FAD of T. thermophilus PRODH is highly solvent-exposed compared with PutA due to a 4-Å shift of helix 8. Structure-based sequence analysis of the PutA/PRODH family led us to identify nine conserved motifs involved in cofactor and substrate recognition. Biochemical studies show that the midpoint potential of the FAD is -75 mV and the kinetic parameters for proline are K_m = 27 mM and k_cat = 13 s^-1. 3,4-Dehydro-L-proline was found to be an efficient substrate, and L-tetrahydro-2-furoic acid is a competitive inhibitor (K_I = 1.0 mM). Finally, we demonstrate that T. thermophilus PRODH reacts with O₂ producing superoxide. This is significant because superoxide production underlies the role of human PRODH in p53-mediated apoptosis, implying commonalities between eukaryotic and bacterial monofunctional PRODHs.

Received for publication, January 31, 2007 , and in revised form, February 28, 2007.

The atomic coordinates and structure factors (code 2G37) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants GM065546 (to J. J. T.) and GM061068 (to D. F. B.), and the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. Part of this research was performed at the Advanced Light Source, which is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Dept. of Energy under Contract DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Chemistry, University of Missouri-Columbia, Columbia, MO 65211. Tel.: 573-884-1280; Fax: 573-882-2754; E-mail: tannerjj{at}missouri.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?