Characterization of a Monomeric Heat-Labile Classical Alkaline Phosphatase from Anabaena sp. PCC7120

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Ming Luo^1,2, Yong-Chao Guo¹, Jiao-Yu Deng¹, Hong-Ping Wei¹, Zhi-Ping Zhang¹, Yan Leng^1,2, Dong Men^1,2, Li-Rong Song³, Xian-En Zhang¹, and Ya-Feng Zhou^1*

¹State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; fax: +86(027)8719-9492; E-mail: zyf@wh.iov.cn; luoming1976@yahoo.cn; guoyc@ust.hk; dengjy@wh.iov.cn; hpwei@wh.iov.cn; zhangzp@wh.iov.cn; x.zhang@wh.iov.cn

²Graduate School, Chinese Academy of Science, Beijing 100039, China; audreyyanleng@yahoo.com.cn; Winter_J_Gates@yahoo.com.cn

³State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; lrsong@ihb.ac.cn

^* To whom correspondence should be addressed.

Received March 12, 2010; Revision received March 30, 2010

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Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (P_i) during P_i starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during P_i starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase AP^A, coded by all2843, is characterized as a metalloenzyme containing Mg²⁺ and Zn²⁺ with molar ratio of 1 : 2. Site-directed mutagenesis analysis indicated that, though the active center of AP^A is highly conserved in comparison with EAP, differences do exist between AP^A and EAP in metal ion coordination. Besides, biochemical analysis revealed that AP^A is a monomeric protein and inactivated rapidly at 50°C. These results suggest that AP^A is the first monomeric heat-labile classical PhoA found in cyanobacteria.

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KEY WORDS: Anabaena sp. PCC7120, phosphorous starvation, alkaline phosphatase, metalloenzyme, site-directed mutagenesis

DOI: 10.1134/S0006297910050172

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