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J. Biol. Chem., Vol. 283, Issue 29, 19981-19990, July 18, 2008

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-Glutamylputrescine Synthetase in the Putrescine Utilization Pathway of Escherichia coli K-12^*

Shin Kurihara¹², Shinpei Oda¹, Yuichi Tsuboi¹, Hyeon Guk Kim, Mayu Oshida, Hidehiko Kumagai, and Hideyuki Suzuki^¶3

From the Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan, Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi-cho, Ishikawa-gun, Ishikawa 921-8836, Japan, and ^¶Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Goshokaido-cho Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan

Glutamate-putrescine ligase (-glutamylputrescine synthetase, PuuA, EC 6.3.1.1 [EC] 1) catalyzes the -glutamylation of putrescine, the first step in a novel putrescine utilization pathway involving -glutamylated intermediates, the Puu pathway, in Escherichia coli. In this report, the character and physiological importance of PuuA are described. Purified non-tagged PuuA catalyzed the ATP-dependent -glutamylation of putrescine. The K_m values for glutamate, ATP, and putrescine are 2.07, 2.35, and 44.6 mM, respectively. There are two putrescine utilization pathways in E. coli: the Puu pathway and the pathway without -glutamylation. Gene deletion experiments of puuA, however, indicated that the Puu pathway was more critical in utilizing putrescine as a sole carbon or nitrogen source. The transcription of puuA was induced by putrescine and in a puuR-deleted strain. The amino acid sequences of PuuA and glutamine synthetase (GS) show high similarity. The molecular weights of the monomers of the two enzymes are quite similar, and PuuA exists as a dodecamer as does GS. Moreover the two amino acid residues of E. coli GS that are important for the metal-catalyzed oxidation of the enzyme molecule involved in protein turnover are conserved in PuuA, and it was experimentally shown that the corresponding amino acid residues in PuuA were involved in the metal-catalyzed oxidation similarly to GS. It is suggested that the intracellular concentration of putrescine is optimized by PuuA transcriptionally and posttranslationally and that excess putrescine is converted to a nutrient source by the Puu pathway.

Received for publication, January 7, 2008 , and in revised form, May 8, 2008.

^* This work was supported in part by the research fund of the Iijima Memorial Foundation (to H. S.). 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.

¹ Supported by the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

² Present address: Japan Collection of Microorganisms, Microbe Division, RIKEN, BioResource Center, Wako, Saitama 351-0198, Japan.

³ To whom correspondence should be addressed. Tel.: 81-75-724-7763; Fax: 81-75-724-7766; E-mail: hideyuki{at}kit.ac.jp.

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