So Anthony K-C, Cot Swan S-W, Espie George S
Department of Botany, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada.Corresponding author; email:
Department of Botany, University of Toronto at Mississauga, 3359 Mississauga Road, Mississauga, Ontario L5L 1C6, Canada.
Funct Plant Biol. 2002 Apr;29(3):183-194. doi: 10.1071/PP01179.
Sequence analysis of the carboxysomal carbonic anhydrase (CcaA) from Synechocystis PCC6803, Synechococcus PCC7942 and Nostoc ATCC29133, indicated high sequence identity to the β class of plant and bacterial carbonic anhydrases (CA), and conservation of the active site region. However, the cyanobacterial enzyme has a C-terminal extension of about 75 amino acids (aa) not found in the plant enzymes, and largely absent from other bacterial enzymes. Using recombinant DNA technology, genes encoding C-terminal truncation products of up to 127 aa were overexpressed in E. coli, and partially purified lysates were analysed for CA-mediated exchange of O between CO2and H2O. Recombinant CcaA proteins with up to 60 aa removed (CcaAΔ60) were catalytically competent, but beyond this there was an abrupt loss of activity. CcaAΔ0, along with CcaAΔ40 and CcaAΔ60, also catalysed the hydrolysis of carbon oxysulfide (COS; an isoelectronic structural analogue of CO2), but CcaAΔ63 and CcaAΔ127 did not, indicating that truncations greater than 62 aa resulted in a general loss of catalytic competency. Analysis of protein-protein interaction using the yeast two-hybrid system revealed that CcaA did not interact with the large or small Rubisco subunits (RbcL and RbcS, respectively) of Synechocystis, but there was strong CcaA-CcaA interaction. This protein interaction also ceased with C-terminal truncations in CcaA greater than 60 aa. The correlation between loss of CcaA-CcaA interaction and CcaA catalytic activity suggests that the proximal portion of the C-terminal extension is required for oligomerization, and that this oligomerization is essential for catalysis by the cyanobacterial enzyme. Thus, the C-terminal extension may play an important role in the function of CA within cyanobacterial carboxysomes, which is not required by the higher plant enzymes.
对来自集胞藻PCC6803、聚球藻PCC7942和念珠藻ATCC29133的羧酶体碳酸酐酶（CcaA）进行序列分析，结果表明其与植物和细菌碳酸酐酶（CA）的β类具有高度的序列同一性，并且活性位点区域保守。然而，蓝藻酶具有约75个氨基酸（aa）的C端延伸，这在植物酶中未发现，并且在其他细菌酶中也基本不存在。利用重组DNA技术，在大肠杆菌中过表达编码长达127个aa的C端截短产物的基因，并对部分纯化的裂解物进行CA介导的CO₂和H₂O之间O交换的分析。去除多达60个aa的重组CcaA蛋白（CcaAΔ60）具有催化活性，但超过此范围则活性突然丧失。CcaAΔ0以及CcaAΔ40和CcaAΔ60也催化了氧硫化碳（COS；CO₂的等电子结构类似物）的水解，但CcaAΔ63和CcaAΔ127则不能，这表明截短超过62个aa会导致催化活性普遍丧失。使用酵母双杂交系统分析蛋白质-蛋白质相互作用表明，CcaA不与集胞藻的大亚基或小亚基Rubisco（分别为RbcL和RbcS）相互作用，但存在强烈的CcaA-CcaA相互作用。当CcaA的C端截短超过60个aa时，这种蛋白质相互作用也会停止。CcaA-CcaA相互作用丧失与CcaA催化活性之间的相关性表明，C端延伸的近端部分是寡聚化所必需的，并且这种寡聚化对于蓝藻酶的催化作用至关重要。因此，C端延伸可能在蓝藻羧酶体中CA的功能中起重要作用，而高等植物酶则不需要。
