CANDIDATE GENES FOR THE PHYCOERYTHROCYANIN ALPHA-SUBUNIT LYASE - BIOCHEMICAL-ANALYSIS OF PECE AND PECF INTERPOSON MUTANTS

The rod substructures of the Anabaena sp, PCC 7120 phycobilisome conta in the light harvesting proteins C-phycocyanin and phycoerythrocyanin (PEG), Even at low light intensities, PEC represents no more than 5% o f the phycobilisome protein, The beta subunits of both proteins carry thioether-linked phycocyanobilin (PCB) at beta-Cys-82 and beta-Cys-155 ; however, C-phycocyanin has PCB at alpha-Cys-84 whereas PEC alpha sub unit carries phycobiliviolin at this position. The Anabaena sp, PCC 71 20 pec operon is made up of five genes, PecB and pecA encode the beta and alpha subunits of PEC, pecC encodes a linker polypeptide associate d with PEC in the rod substructure, and pecE and pecF are genes of unk nown function that show a high degree of homology to cpcE and cpcF, th at encode a C-phycocyanin alpha subunit PCB lyase (Fairchild, C, D., Z hao, J., Zhou, J., Colson, S, E., Bryant, D. A., and Glazer, A. N. (19 92) Proc. Natl. Acad. Sci. U.S. A. 89, 7017-7021), Insertional mutants in pecE and pecF, and an interposon mutant in which a portion of both pecE and pecF was deleted, were constructed. All three types of mutan ts grew 1.3 times slower than wild-type under limiting light condition s and showed a 20% reduction in the PCB content of whole cells relativ e to chlorophyll alpha. Holo-PEC was missing from the phycobilisomes o f all three types of mutants and the level of the PEC linker polypepti de was reduced relative to the wild-type. However, similar to 30% of t he wild-type level of the PEC beta subunit was present in all of these phycobilisomes, In contrast, the PEC alpha subunit was barely detecta ble in the pecE and pecF mutants, but was present in the pecEF deletio n mutant as a PCB-adduct in a 1:1 ratio with the PEC beta subunit. The identity of this ''unnatural'' adduct was confirmed by isolation of t he subunit and amino-terminal sequencing, These biochemical results su pport the inference that pecE and pecF encode a PEC a subunit phycobil iviolin lyase, and, in conjunction with earlier findings, demonstrate that phycobiliprotein bilin lyases show high selectivity (rather than absolute specificity) for both the bilin and the polypeptide substrate .