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J. Biol. Chem., Vol. 281, Issue 13, 8573-8581, March 31, 2006

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Chromophore Attachment to Phycobiliprotein -Subunits

PHYCOCYANOBILIN:CYSTEINE-84 PHYCOBILIPROTEIN LYASE ACTIVITY OF CpeS-LIKE PROTEIN FROM ANABAENA Sp. PCC7120^*

Kai-Hong Zhao¹, Ping Su, Jian Li, Jun-Ming Tu, Ming Zhou, Supported by the National Natural Science Foundation of China (Grant 30540070)², Claudia Bubenzer^||, and Hugo Scheer, Supported by the Deutsche Forschungsgemeinschaft (Grant SFB 533, TPA1)^||13

From the Colleges of Life Science and Technology and of Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, Hubei, P.R. China and the ^||Department Biologie I, Universität München, Menzinger Strasse 67, D-80638 München, Germany

The gene alr0617, from the cyanobacterium Anabaena sp. PCC7120, which is homologous to cpeS from Gloeobacter violaceus PCC 7421, Fremyella diplosiphon (Calothrix PCC7601), and Synechococcus sp. WH8102, and to cpcS from Synechococcus sp. PCC7002, was overexpressed in Escherichia coli. CpeS acts as a phycocyanobilin: Cys-84-phycobiliprotein lyase that can attach, in vitro and in vivo, phycocyanobilin (PCB) to cysteine-84 of the apo--subunits of C-phycocyanin (CpcB) and phycoerythrocyanin (PecB). We found the following: (a) In vitro, CpeS attaches PCB to native CpcB and PecB, and to their C155I-mutants, but not to the C84S mutants. Under optimal conditions (150 mM NaCl and 500 mM potassium phosphate, 37 °C, and pH 7.5), no cofactors are required, and the lyase had a K_m(PCB) = 2.7 and 2.3µM, and a k_cat = 1.7 x 10^-5 and 1.1 x 10^-5 s^-1 for PCB attachment to CpcB (C155I) and PecB (C155I), respectively; (b) Reconstitution products had absorption maxima at 619 and 602 nm and fluorescence emission maxima at 643 and 629 nm, respectively; and (c) PCB-CpcB(C155I) and PCB-PecB(C155I), with the same absorption and fluorescence maxima, were also biosynthesized heterologously in vivo, when cpeS was introduced into E. coli with cpcB(C155I) or pecB(C155I), respectively, together with genes ho1 (encoding heme oxygenase) and pcyA (encoding PCB:ferredoxin oxidoreductase), thereby further proving the lyase function of CpeS.

Received for publication, December 27, 2005 , and in revised form, January 30, 2006.

^* 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 Table S1 and supplemental Figs. S1-S3.

¹ Supported by a Volkswagen Stiftung Partnership (I/77900).

²To whom correspondence may be addressed. Tel./Fax: 86-27-8754-1634; E-mail: khzhao{at}163.com. ³To whom correspondence may be addressed. Tel.: 49-89-17861-295; Fax: 49-89-17861-271; E-mail: hugo.scheer{at}lmu.de.

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