Research Communication Identification and Biochemical Properties of Dps (starvation-induced DNA binding protein) from Cyanobacterium Anabaena sp. PCC 7120 Xiong Wei, He Mingjia, Li Xiufeng, Gao Yang and Wu Qingyu Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, PR China Summary DNA-binding Proteins from Starved cells (Dps) are anti-stress iron proteins preserving bacteria from oxidative damage. Based on sequence alignment, a 564-bp open reading frame (all1173) encoding product in Anabaena sp. PCC 7120 shared high similarity with Dps family proteins. RT-PCR showed all1173 is active at transcriptional level in Anabaena sp. PCC 7120 cells. We accordingly cloned the all1173 into prokaryotic expression system, purified the corresponding recombinant protein (Dps1173) and characterized its properties in vitro. According to CD spectrum and non-denaturing electrophoresis assays, recombinant Dps1173 was alpha helix riched, and was likely to form dodecametric oligomer under native conditions. Fluorescence titration experiment revealed two major iron binding sites within Dps1173 monomer, indicating its potential ferroxidase activity. Although phenomena of direct DNA binding was not observed in Electrophoretic mobility shift assay, Dps1173 could also protect DNA from H 2 O 2 stress for its iron scavenging capacity. This is the first description of Dps from heterocystous cyanobacterium Anabaena sp. PCC 7120. IUBMB Life, 59: 675–681, 2007 Keywords Anabaena sp. PCC 7120; DNA protection; Dps; ferrox- idase; iron detoxification. INTRODUCTION DNA-binding Proteins from Starved cells (Dps) are a class of iron protein throughout wide ranges of microorganisms such as Escherichia coli (1), Agrobacterium tumefaciens (2), Listeria innocua (3) and Bacillus anthracis (4), playing dual roles in both oxidative stress defense and iron bioavailability. In E. coli, the structure, biochemical characterization and cellular regulation of Dps protein have been extensively investigated (5–9). E. coli Dps, first characterized as an antioxidant protein, was abundantly expressed during the stationary phase in response to nutrient stress or oxidative stress (10). It was shown to incorporate DNA without sequence specificity and further exert protection against hydrogen peroxide stress, Dnase cleavage, UV radiation and acid shock (10, 11). Based on sequence analysis, Dps are endowed with conserved ferroxidase domain, implicating that they also belong to ferritin family. Following structural and related researches (12, 13) further illuminated that Dps proteins are able to capture up to hundreds of free iron atoms and convert them into available forms, thereby guaranteeing continuous supply of iron to microorganisms. The heterocystous cyanobacterium Anabaena sp. PCC 7120, a model organism for the study of cell division and nitrogen fixation, are inevitably undergone iron limitation, oxidative stress or other growth-limiting conditions in natural aquatic environment. It is worthy of noting that iron implicates in nitrogen fixing as a key cofactor because nitrate reduction and nitrogen fixation require numerous iron- containing enzymes (14). Iron starvation was indicated to cause an increasing production of Reactive Oxygen Species in Anabaena and disturb regular physiological functions (15). As a result, Anabaena cells have developed efficient regulatory mechanisms that enable themselves to transport, store and release iron in response to iron restriction. By searching the genome database of Anabaena sp. PCC 7120, the deduced ORF for encoding Dps (all1173) was revealed and shown high identity with other identified Dps encoding genes. However, the nature of this putative protein, especially its potential role in iron metabolism and antioxida- tion in Anabaena cells is still unclear. In the present study, we report on the identification and purification of Anabaena all1173, demonstrating its iron incorporation and DNA protection capacity in vitro. Received 18 June 2007; accepted 1 August 2007 Address correspondence to: Wu Qingyu, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China. Tel/Fax: þ86 10 62781825. E-mail: qingyu@mail.tsinghua.edu.cn IUBMB Life, 59(10): 675 – 681, October 2007 ISSN 1521-6543 print/ISSN 1521-6551 online Ó 2007 IUBMB DOI: 10.1080/15216540701606926