CONICET | Buscador de Institutos y Recursos Humanos

Proteinengineering by the addition of substrate binding domains is becoming a widelyused strategy to improve enzyme properties. These binding domains are oftenused to increase the catalytic efficiency, as affinity tags to facilitateprotein purification and also for targeting a protein to specific cellularlocations. CBMs (carbohydrate binding modules) arenon-catalytic protein domains that are naturally present insome enzymes and are associated with the ability to bind polysaccharides. AmongCBMs, there is a subgroup called SBDs that have an evolutionary advantage dueto the presence of two starch binding sites. The mechanisms of action of CBMs differand are characteristic of the enzyme in which they are present. They can act bybringing the catalytic domain closer to the substrates, as a scaffold forprotein-protein interaction and, additionally, they can break the structure ofsubstrates increasing the catalytic efficiency of the enzyme. The latter isparticularly important when the substrates are structured, such as starchgranules or plant cell walls. Typically,the fusion of CBMs in cis to generate chimeric proteins is used to evaluateconstructs as possible biotechnological tools. Our laboratory has demonstratedthe interaction of SBDs in cis with the glycogen synthase (GS) from Agrobacteriumtumefaciens. The addition of the D3 domain from A.thaliana SSIII to the GS conferred a higher capacity for glycogen biosynthesis,suggesting that the careful design of fusion proteins can led to the productionof a fully active and conformationally stable molecule composed of domains thatbelong to different kingdoms, in this case, plants and bacteria. The presenceof a polysaccharide-binding site outside the active site of the enzyme wouldlead to improve the binding capacity through multiple contacts, increasing thelocal concentration of non-reducing ends in the active site and resulting in agreater processivity of the enzyme. While there are many studies on the effect of CBMs in cis, fewstudies have been conducted to evaluate their effect in trans. We analyzedhere the ability of different CBMs, coming from different families (a CBM20from a Ostreococcus tauri protein (CBM20CP) and three xylan binding domains(XYL1-3, classified in the CBM22 family) from a xylanase from A. thaliana(AtXyn1) to act in trans on two commercial enzymes, an amylase (AmyC) and a xylanase(XYNA) respectively. The CBM20 is located in the central position of a proteinwithout associated catalytic activity. Therefore, we evaluated the effect of therecombinant CBM20 and the full CBM20CP. We found that the addition of CBM20 hada little impact on the activity of AmyC, while the addition of the full proteinsignificantly increased (about 90%) the catalytic efficiency of the enzyme. Onthe other hand, of the three CBMs from AtXyn1 used, only XYL1 was able toincrease the Vmax of XYNA by 70% without significant differences in the Km,suggesting that the increase in the catalytic efficiency is exclusively due tothe increase in Vmax and not in its affinity for the substrate. Our resultsshow that the addition in trans of CBM20CP and XYL1 would be a useful strategyto improve the activity of some enzymes that use different polysaccharides assubstrates.

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