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

J. Biol. Chem., Vol. 278, Issue 52, 53105-53111, December 26, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/52/53105    most recent M304408200v2 M304408200v1 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Yoshimura, T. Articles by Shimizu, T. Search for Related Content PubMed PubMed Citation Articles by Yoshimura, T. Articles by Shimizu, T.

Relationships between Heme Incorporation, Tetramer Formation, and Catalysis of a Heme-regulated Phosphodiesterase from Escherichia coli

A STUDY OF DELETION AND SITE-DIRECTED MUTANTS^*

Tokiko Yoshimura, Ikuko Sagami, Yukie Sasakura, and Toru Shimizu

From the Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (N147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg²⁺ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe²⁺ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.

Received for publication, April 28, 2003 , and in revised form, October 3, 2003.

^* 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.

To whom correspondence should be addressed: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Tel.: 81-22-217-5604/5605; Fax: 81-22-217-5604/5390; E-mail: shimizu{at}tagen.tohoku.ac.jp.

This article has been cited by other articles:

				T. Yoshimura-Suzuki, I. Sagami, N. Yokota, H. Kurokawa, and T. Shimizu DOSEc, a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli J. Bacteriol., October 1, 2005; 187(19): 6678 - 6682. [Abstract] [Full Text] [PDF]

				H. Kurokawa, D.-S. Lee, M. Watanabe, I. Sagami, B. Mikami, C. S. Raman, and T. Shimizu A Redox-controlled Molecular Switch Revealed by the Crystal Structure of a Bacterial Heme PAS Sensor J. Biol. Chem., May 7, 2004; 279(19): 20186 - 20193. [Abstract] [Full Text] [PDF]

				S. Taguchi, T. Matsui, J. Igarashi, Y. Sasakura, Y. Araki, O. Ito, S. Sugiyama, I. Sagami, and T. Shimizu Binding of Oxygen and Carbon Monoxide to a Heme-regulated Phosphodiesterase from Escherichia coli: KINETICS AND INFRARED SPECTRA OF THE FULL-LENGTH WILD-TYPE ENZYME, ISOLATED PAS DOMAIN, AND MET-95 MUTANTS J. Biol. Chem., January 30, 2004; 279(5): 3340 - 3347. [Abstract] [Full Text] [PDF]