Báo cáo khoa học: Starch-binding domains in the CBM45 family – low-affinity domains from glucan,

Starch-binding domains in the CBM45 family – low-affinity

domains from glucan, water dikinase and a-amylase

involved in plastidial starch metabolism

Mikkel A. Glaring1,2, Martin J. Baumann1

, Maher Abou Hachem1

, Hiroyuki Nakai1

, Natsuko Nakai1

,

Diana Santelia3

, Bent W. Sigurskjold4

, Samuel C. Zeeman3

, Andreas Blennow2 and Birte Svensson1

1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Kongens Lyngby, Denmark

2 VKR Research Centre Pro-Active Plants, Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of

Copenhagen, Frederiksberg, Denmark

3 Department of Biology, ETH Zu¨rich, Switzerland

4 Department of Biology, University of Copenhagen, Denmark

Introduction

Starch is deposited as water-insoluble granules in the

amyloplasts of tubers, roots and seeds for long-term

storage, and in the chloroplasts of green tissues during

the day as short-term storage for the following night

(transitory starch). The granules are composed of two

different polymers of glucose linked via a-(1,4)-glyco￾sidic bonds: amylose, which is essentially linear, and

amylopectin, which also contains a-(1,6)-glycosidic

Keywords

carbohydrate-binding module; starch

metabolism; starch-binding domain;

a-amylase; a-glucan, water dikinase

Correspondence

B. Svensson, Enzyme and Protein

Chemistry, Department of Systems Biology,

Technical University of Denmark, Søltofts

Plads, Building 224, DK-2800 Kongens

Lyngby, Denmark

Fax: +45 45886307

Tel: +45 45252740

E-mail: [email protected]

(Received 17 November 2010, revised 5

January 2011, accepted 31 January 2011)

doi:10.1111/j.1742-4658.2011.08043.x

Starch-binding domains are noncatalytic carbohydrate-binding modules

that mediate binding to granular starch. The starch-binding domains from

the carbohydrate-binding module family 45 (CBM45, http://www.cazy.org)

are found as N-terminal tandem repeats in a small number of enzymes,

primarily from photosynthesizing organisms. Isolated domains from repre￾sentatives of each of the two classes of enzyme carrying CBM45-type

domains, the Solanum tuberosum a-glucan, water dikinase and the Arabid￾opsis thaliana plastidial a-amylase 3, were expressed as recombinant pro￾teins and characterized. Differential scanning calorimetry was used to

verify the conformational integrity of an isolated CBM45 domain, reveal￾ing a surprisingly high thermal stability (Tm of 84.8 C). The functionality

of CBM45 was demonstrated in planta by yellow ⁄ green fluorescent protein

fusions and transient expression in tobacco leaves. Affinities for starch and

soluble cyclodextrin starch mimics were measured by adsorption assays,

surface plasmon resonance and isothermal titration calorimetry analyses.

The data indicate that CBM45 binds with an affinity of about two orders

of magnitude lower than the classical starch-binding domains from extra￾cellular microbial amylolytic enzymes. This suggests that low-affinity

starch-binding domains are a recurring feature in plastidial starch metabo￾lism, and supports the hypothesis that reversible binding, effectuated

through low-affinity interaction with starch granules, facilitates dynamic

regulation of enzyme activities and, hence, of starch metabolism.

Abbreviations

AMY3, a-amylase 3; AtAMY3, Arabidopsis thaliana a-amylase 3; CBM, carbohydrate-binding module; DSC, differential scanning calorimetry;

GWD, a-glucan, water dikinase; IPTG, isopropyl thio-b-D-galactoside; ITC, isothermal titration calorimetry; PWD, phosphoglucan, water

dikinase; SBD, starch-binding domain; SPR, surface plasmon resonance; StGWD, Solanum tuberosum a-glucan, water dikinase;

TEV, tobacco etch virus; YFP ⁄ GFP, yellow ⁄ green fluorescent protein.

FEBS Journal 278 (2011) 1175–1185 ª 2011 The Authors Journal compilation ª 2011 FEBS 1175