oligomeric structure, enzyme kinetics, and substrate specificity of
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THE JOURNAI. OF BloLoGlrnL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 269, No. 12, Issue of March 25, pp. 8686-8694, 1994 Printed in U.S.A.
Oligomeric Structure, Enzyme Kinetics, and Substrate Specificity of the Phycocyanin a Subunit Phycocyanobilin Lyase*
(Received for publication, October 12, 1993, and in revised form, December 28, 1993)
Craig D. Fairchild4 and Alexander N. Glazers From the Department of Molecular and Cell Biology, Uniuersity of California, Berkeley, California 94720
Phycobiliproteins carry linear tetrapyrrole chromo- phores (bilins) thioether-linked to specific cysteine resi- dues. The process of bilin attachment to apoprotein in vivo has been characterized for only one bilin attach- ment site on one phycobiliprotein, that on the a subunit of phycocyanin (apc). In the cyanobacterium Synecho- coccus sp. PCC 7002, the addition of phycocyanobilin to apo-aPC is catalyzed by the protein products of the cpcE and cpcF genes. We have purified and further character- ized the recombinant CpcE and CpcF proteins. CpcE and CpcF form an enzymatically active 1:l complex (Cp- cEF), stable to size exclusion chromatography. CpcEF causes a reduction in apc fluorescence and strongly af- fects its absorption spectrum but has no effect on the p subunit. The CpcEF bilin addition activity exhibits simple Michaelis-Menten kinetics with respect to the apo-aPC and to bilin. CpcEF also catalyzes the addition of phycoerythrobilin to apo-aPc; phycoerythrobilin is thought to be on the biosynthetic pathway of phycocya- nobilin. CpcEF shows a preference for phycocyanobilin relative to phycoerythrobilin, both in binding affinity and in the rate of catalysis, sufficient to account for selective attachment of phycocyanobilin to apo-aPC.
In cyanobacteria and red algae, phycobiliproteins are the major components of a thylakoid membrane-associated macro- molecular light-harvesting complex, the phycobilisome (1). The phycobiliproteins carry covalently attached linear tetrapyrrole prosthetic groups (bilins). In different species, the number of different bilins on the various phycobiliproteins ranges from one to three, and the number of distinct attachment sites ranges from eight to more than 20.
The bilins phycocyanobilin (PCB) and phycoerythrobilin (PEB) can be cleaved from phycobiliproteins by refluxing in methanol (2, 3). This treatment results in the elimination of a phycobiliprotein cysteine from the 3 carbon of the bilin to yield a linear tetrapyrrole with an ethylidene at the C-3 position (the IUPAC numbering scheme for bilins and the structures of PCB and PEB is shown in Ref. 4).
The conformation and interactions of bilins with the poly-
Grant GM 28994 (to A. N. G.). The costs of publication of this article * This work was supported in part by National Institutes of Health
were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Present address: University of California BerkeleylLTSDA Plant Gene Expression Center, 800 Buchanan St., Albany, CA 94710.
(i To whom correspondence should be addressed: MCB:Stanley/
94720. Tel.: 510-642-3126; Fax: 510-643-9290. Donner ASU, 229 Stanley Hall, University of California, Berkeley, CA
erythrobilin; cypc, C-phycocyanin cy subunit; DTT, dithiothreitol; PAGE, The abbreviations used are: PCB, phycocyanobilin; PEB, phyco-
polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; PCB*, addition-competent form of PCB; SEC, size exclusion chromatography; MBV, mesobiliverdin.
peptide within native phycobiliproteins greatly affect their spectroscopic properties. Generally, a native phycobiliprotein has a strongly enhanced long wavelength visible absorption peak and a much higher fluorescence quantum yield relative to its denatured form or to bilin not associated with protein.
Phycocyanin is a phycobiliprotein that bears adducts of PCB a t residues a-Cys-84 and p-Cys-82 and p-Cys-155. PCB reacts spontaneously with apophycocyanin in vitro at a-Cys-84 and p-Cys-82 (but not a t p-155), to form an unnatural adduct, 3- cysteinylmesobiliverdin, which contains an extra double bond between carbons 2 and 3 of ring A of the bilin (5, 6). PEB also reacts at a-Cys-84 and 6-Cys-82 to form a similar adduct with a double bond at C2-C3 of ring A (7).
In the cyanobacterium Synechococcus sp. PCC 7002, the at- tachment of PCB to the a subunit of phycocyanin (apc) is cata- lyzed by a specific protein bilin lyase, which is encoded by the genes cpcE and cpcF. The phycobiliproteins of this organism have eight PCB attachment sites on seven polypeptides. Inac- tivation of cpcE or cpcF by interposon insertion leads to loss of correct bilin attachment to apc but not to any of the other PCB attachment sites (8, 9). The protein products of these genes, CpcE and CpcF, have been expressed separately in Escherichia coli and shown to catalyze together the correct addition of PCB to apo-aPc (10). A number of genes with varying degrees of homology to cpcE and cpcF have been characterized in other cyanobacteria (for a review see Ref. ll), although none of these has been shown to encode a protein bilin lyase.
The present study presents further characterization of CpcE and CpcF and of their enzymatic activity. Purification schemes for recombinant CpcE and CpcF are presented. The purified proteins were used to establish that CpcE and CpcF form a 1:1 complex, which is henceforth termed CpcEF. Various assays for catalysis and substrate binding failed to show any activity for the individual CpcE or CpcF polypeptides.
Since PEB is a biosynthetic intermediate in the formation of PCB in a red alga (12), and presumably in cyanobacteria as well, the specificity of CpcEF for its bilin substrate is of par- ticular interest. Kinetic data presented here show that CpcEF, although it can catalyze the addition of PEB to apo-aPC, exhib- its specificity for PCB both in binding afflnity and the rate of catalysis.
CpcEF has also been shown to catalyze the rapid transfer of bilin from holo-aPC to apo-aPc (10). This result implies that Cp- cEF can access the thioether linkage between protein and bilin in apc, which should require at least partial unfolding of the holo subunit. In support of this notion, we report here that CpcEF alters the absorption and dramatically reduces the fluorescence emission of the a subunit, but not the p subunit, of phycocyanin. A change in the fluorescence of a holo subunit may be a conve- nient assay for phycobiliprotein bilin lyases yet to be discovered.
EXPERIMENTAL PROCEDURES CpcE and CpcFPurification-Crude CpcE and CpcF inclusion bodies
were prepared and solubilized in 9 M urea at pH 1.9, as described
Phycocyanin a Subunit Phycocyanobilin Lyase 8687
previously (10). The protein solution was brought to 10 mM DTT and pH 7.5-8.0 by the addition of solid DTT and 1.5 M Tris-HC1, pH 8, and incubated at room temperature for 1 h. CpcE and CpcF solutions were then ultracentrifuged (30 min at 100,000 x g) and filtered through a 0.2-pm pore size membrane. Each solution (4-5 ml) was then exchanged into 8 M urea, 20 mM Tris-HCI, pH 7.5 (plus 1 nm DTT for CpcF) by passage through a Sephadex G-25 column (80-ml bed volume) in the same solvent.
Aliquots of each protein (9 ml, 35-40 mg of protein) were loaded onto a Mono Q HR 10/10 column (Pharmacia LKB Biotechnology, Inc.) on a Perkin-Elmer series 410 Bio LC system; the column was preequili- brated in 8 M urea, 20 nm Tris-HC1, pH 7.5, and the flow rate through- out was 4 mumin. The column was washed for 2 min in the starting solvent and then eluted with a linear gradient of NaCl in the same solvent: 35 min, 0-0.35 M NaCl for CpcE; 35 min, 0-0.2 M NaCl for CpcF. Two-ml fractions were collected. The fractions were evaluated by SDS- PAGE, and those containing CpcE or CpcF were pooled, brought to 5 mM DTT, and acidified to pH 2.5 with concentrated HCI.
Reverse-phase HPLC was performed with a semipreparative scale C, column (Hi-Pore RP-304, Bio-Rad) in the solvent system used for the separation of phycobiliprotein subunits (13): aqueous, 0.1% trifluoro- acetic acid; organic, 2:l acetonitri1e:isopropyl alcohol, 0.1% trifluoroace- tic acid. Aliquots of CpcE (1.4 ml, 2 mg of protein) and CpcF (1.5 ml, 1.5 mg of protein) Mono Q pools were loaded without further treatment onto the C, column in 35% organic solvent, 3 mumin flow rate. The elution gradients, after 2 min at 35% organic solvent, were: CpcE, 3 5 4 5 % organic in 2 min, 5.571% in 32 min; CpcF, 3 5 4 5 % organic in 5 min, 5540% in 5 min, and 60-70% in 30 min.
CpcE and CpcF pools were reduced to 0.20 volume by rotary evapo- ration. Some precipitate formed during this step; this precipitate dis- solved on dilution of the concentrated pools 1:2 (v/v) with 9 M urea-HCI, 10 m Tris-HC1, pH 2.5. The pools were dialyzed against the urea diluent containing 1 nm DTT. Dialyzed CpcE and CpcF were stored at 4 "C (DTT was added to the CpcF solution to a final concentration of 5 mM).
Renaturation of CpcE and CpcF-For assay of fractions from chro- matography, CpcE or CpcF solutions in urea (HPLC fractions were reduced to 0.20 volume by evaporation with a stream of N, and diluted 1:5 with 9 M acid-urea) were dilu