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Phycocyanobilin Synthesis in the Unicellular Rhodophyte Cyanidium Caldarium by ROBERT F

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Phycocyanobilin Synthesis in the Unicellular Rhodophyte Cyanidium Caldarium by ROBERT F Biochem. J. (1978) 172, 569-576 569 Printed in Great Britain Phycocyanobilin Synthesis in the Unicellular Rhodophyte Cyanidium caldarium By ROBERT F. TROXLER Department ofBiochemistry, Boston University School of Medicine, Boston, MA 02118, U.S.A. and PATRICK KELLY School of Chemistry, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K. and STANLEY B. BROWN Department ofBiochemistry, University ofLeeds, 9 Hyde Terrace, Leeds LS2 9LS, U.K. (Received 10 October 1977) Light is required for synthesis of the accessory photosynthetic pigment phycocyanin in cells of the unicellular rhodophyte Cyanidium caldarium. Phycocyanin is a conjugated protein composed of polypeptide subunits to which the light-absorbing bile pigment chromophore phycocyanobilin is covalently attached. Dark-grown cells of C. caldarium are unable to make phycocyanin, but when incubated in the dark with 5-aminolaevulinate the cells synthesize and excrete a protein-free phycobilin (algal bile pigment) into the suspending medium. The electronic absorption spectrum, electron impact mass spectrum, chromatographic properties and imide products obtained after chromic acid degradation of the excreted phycobilin were identical with those of phycocyanobilin cleaved from phycocyanin in boiling methanol. This establishes the structural identity between the excreted phycobilin, which is the end product of bile-pigment synthesis in vivo, and the chromophore cleaved from phycocyanin in boiling methanol. The significance of the structure of the excreted phycobilin with respect to the events surrounding the assembly of the phycocyanin molecule in vivo is discussed. Phycobiliproteins are accessory pigments found in lished (Chapman, 1973; O'Carra & hEocha, the photosynthetic apparatus of red (Rhodophyta), 1976). The classic studies by Lemberg (1928, 1930) blue-green (Cyanophyta) and cryptomonad (Crypto- demonstrated that algal bile pigments were released phyta) algae (O'Carra & hEocha, 1976). Allo- from phycobiliproteins in hot concentrated acid and phycocyanin, phycocyanin and phycoerythrin are the alkali, but, recognizing that the hydrolytic conditions principal phycobiliproteins in these organisms yielded bile-pigment artifacts, Lemberg (1928, 1930) (Bogorad, 1975). Phycocyanobilin is the light- referred to the native chromophores of phycocyanin absorbing chromophore of allophycocyanin and and phycoerythrin as phycocyanobilin and phyco- phycocyanin, and phycoerythrobilin is the major erythrobilin respectively. More recently, phycobilins chromophore of phycoerythrin (Bennett & Siegel- have been cleaved from apoproteins by hydrolysis in man, 1977). Phycocyanobilin and phycoerythrobilin concentrated acid at room temperature (O hEocha, are linear tetrapyrroles structurally related to mam- 1963; Rudiger & O'Carra, 1969) or by boiling malian bile pigments (Siegelman et al., 1968; Troxler, phycobiliproteins in methanol (Fujita & Hattori, 1977). Algal bile pigments (phycobilins) are syn- 1963; Cole et al., 1968; Siegelman et al., 1968; thesized from 5-aminolaevulinate via the porphyrin- Troxler, 1972). 0 hEocha (1963) showed that the synthetic pathway and ultimately arise from the phycobilin obtained by hydrolysis of phycocyanin in carbon skeleton of protoporphyrin IX, i.e. haem concentrated HCl at room temperature was spectrally (Troxler, 1972). Phycocyanobilin is derived from its identical with the covalently attached chromophore porphyrin precursor by a mechanism (Troxler & of phycocyanin in 8 M-urea, and he regarded the Brown, 1977) identical with that used for haem cleaved phycobilin as phycocyanobilin. This phyco- conversion into bile pigment in mammals (Brown & bilin has not been crystallized and its structure is not King, 1975). known (O'Carra & hEocha, 1976). The phycobilin Phycobilins are covalently linked to apoproteins in isolated from phycocyanin by boiling methanol has algal cells, but the exact nature of the phycobilin- been purified as the dimethyl ester derivative, and its apoprotein linkages has not been definitively estab- structure has been determined by degradative tech- Vol. 172 570 R. F. TROXLER, P. KELLY AND S. B. BROWN CO2H CO2H CH3 CH2 CH2 CH3 IlI CH3 CH CH3 CH2 CH2 CH3 CH3 CH2 H - IIII III IV 0 CN C N C,N C N H H H H H H Phycocyanobilin CO2H CH3 CH2 CH3 CH3 CH CH3 CH2 CH3 CH2 H H N 0~~ N 0 N H H H Ethylidinemethylsuccinimide Haematinic acid Ethylmethylmaleimide Fig. 1. Structures ofphycocyanobilin and imides derived from phycocyanobilin The imides were obtained by degradation of phycocyanobilin in 1% (w/v) CrO3/lM-H2SO4 at 100°C for 30min (see the text). niques (Rudiger, 1968; Rudiger & O'Carra, 1969), grown in the dark. Dark-grown cells synthesize mass spectrometry and n.m.r. spectroscopy (Cole photosynthetic pigments, however, when placed et al., 1968; Crespi et al., 1968; Schram & Kroes, in the light. C. caldarium mutant III-D-2 was used 1971; Beuhler et al., 1976). This phycobilin has been in the present investigation because this strain considered in some laboratories to be synonymous produces more pigment per cell in the light than with phycocyanobilin (Fig. 1) (Siegelman et al., does the wild-type (Troxler & Bogorad, 1966). 1968; Troxler, 1972; Beuhler et al., 1976) and is referred to as such below. Culture conditions Troxler & Bogorad (1966) found that cells of the Algal cells were grown from a small inoculum on a unicellular rhodophyte Cyanidium caldarium excreted rotary shaker in 1500ml of medium supplemented a protein-free phycobilin into the growth medium with 1 % (w/v) glucose (Allen, 1959) in 3-litre when incubated with 5-aminolaevulinate, the first Fernbach flasks at 370C in the dark for 7-10 days committed intermediate in the porphyrin pathway. (standard conditions). Dark-grown cells were sub- The excreted phycobilin was considered to be the sequently treated in one of two ways. (a) Dark-grown linear tetrapyrrole (i.e. bile pigment), which becomes algal cells were collected by centrifugation (15OOOg covalently attached to apoprotein during phyco- for 15min), resuspended in medium (I500ml) minus cyanin biosynthesis in vivo (Troxler & Lester, 1967; glucose and 5-aminolaevulinate, and incubated for Troxler, 1972). 72h under standard conditions in the light (General In the present paper, the properties of the excreted Electric fluorescent tubes, F17PG, CW; approx. phycobilin are compared with those of phycocyano- 54001x). The cells produced chlorophyll a and bilin cleaved from phycocyanin by boiling methanol, phycobiliproteins in the light. Phycocyanin was and the relationship between the excreted phycobilin isolated and purified from these pigmented cells as and phycocyanin biosynthesis in vivo is discussed. described below. (b) Algal cells were collected by centrifugation at 15000g for 5min, and 1 ml of Experimental packed cells was placed in 9ml of medium containing 1 % glucose and 50mM-5-aminolaevulinate (Sigma Organism Chemical Co., St. Louis, MO, U.S.A.), and several Cyanidium caldarium (Allen, 1959) is a unicellular such suspensions were incubated in 25 ml Erlenmeyer rhodophyte that synthesizes chlorophyll a, allo- flasks for 72h in the dark. The phycobilin synthesized phycocyanin and phycocyanin in the light, but is from 5-aminolaevulinate under these conditions was unable to make these photosynthetic pigments when isolated and purified as described below. 1978 ALGAL BILE-PIGMENT BIOSYNTHESIS 571 Preparation ofphycocyanin College, PA, U.S.A.) was added and the solution was After illumination, algal cells were collected by refluxed for 2 min. The solution was cooled to room centrifugation at 15000g for 5min, resuspended in temperature, 50ml of water was added and the algal 0.1 M-phosphate buffer (KH2PO4/K2HPO4), pH 7.0 bile-pigment diesters were extracted into chloroform. (1 ml of cells/10ml of buffer), and disrupted by sonica- The chloroform solution was washed with water, tion with a Branson sonic oscillator (model W 185) filtered through chloroform-moistened filter paper, for 10min as described by Brown & Troxler (1977). evaporated to dryness under a stream of N2 and the Particulate matter was removed by centrifugation at product stored at -20°C until used. 36000g for 30min and the resulting supernatant was made 50% saturated with (NH4)2SO4. The pre- Thin-layer chromatography cipitated phycocyanin (and small quantities of allo- The bile-pigment dimethyl esters were dissolved in phycocyanin) were collected by centrifugation, chloroform, applied to 20cm x 20cm silica-gel dialysed overnight against water (50 vol.) at 4°C and plates (Absorbosil-5; Applied Science Laboratories) the dialysed samples were centrifuged at 1000OOg and chromatographed in one of the following solvent for 1h to remove traces of chlorophyll a. Phyco- systems (Cole et al., 1968): (1) methylene dichloride/ cyanin in the supernatant was purified by ion- ethyl acetate (4:1, v/v); (2) benzene/ethanol (9:1, exchange chromatography on brushite columns v/v); (3) carbon tetrachloride/methyl acetate (2:1, (2.5 cm x 20cm) developed with potasium phosphate v/v). The band of purified bile-pigment diester was buffers (pH 7.0) of increasing ionic strength as scraped off the chromatogram, eluted from silica gel described by Brown & Troxler (1977). Phycocyanin, with chloroform or ethanol, which was evaporated to which was eluted from the columns in 0.01-0.025M- dryness, and the methylated bile pigments were stored potassium phosphate buffer, was precipitated by at -20°C until used. adding solid (NH4)2SO4 to 80% saturation, and the precipitate was dialysed overnight
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