The characterization of modified starch branching enzymes: toward the control of starch chain-length distributions

Cheng Li; Alex Chi Wu; Rob Marc Go; Jacob Malouf; Mark S Turner; Alpeshkumar K Malde; Alan E Mark; Robert G Gilbert

doi:10.1371/journal.pone.0125507

The characterization of modified starch branching enzymes: toward the control of starch chain-length distributions

PLoS One. 2015 Apr 13;10(4):e0125507. doi: 10.1371/journal.pone.0125507. eCollection 2015.

Authors

Cheng Li¹, Alex Chi Wu¹, Rob Marc Go², Jacob Malouf², Mark S Turner³, Alpeshkumar K Malde⁴, Alan E Mark⁴, Robert G Gilbert¹

Affiliations

¹ Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, Hubei, China; Centre for Nutrition and Food Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, Australia.
² Centre for Nutrition and Food Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, Australia.
³ Centre for Nutrition and Food Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD, Australia; School of Agriculture and Food Sciences, The University of Queensland, Brisbane, QLD, Australia.
⁴ School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia.

Abstract

Starch is a complex branched glucose polymer whose branch molecular weight distribution (the chain-length distribution, CLD) influences nutritionally important properties such as digestion rate. Chain-stopping in starch biosynthesis is by starch branching enzyme (SBE). Site-directed mutagenesis was used to modify SBEIIa from Zea mays (mSBEIIa) to produce mutants, each differing in a single conserved amino-acid residue. Products at different times from in vitro branching were debranched and the time evolution of the CLD measured by size-exclusion chromatography. The results confirm that Tyr352, Glu513, and Ser349 are important for mSBEIIa activity while Arg456 is important for determining the position at which the linear glucan is cut. The mutant mSBEIIa enzymes have different activities and suggest the length of the transferred chain can be varied by mutation. The work shows analysis of the molecular weight distribution can yield information regarding the enzyme branching sites useful for development of plants yielding starch with improved functionality.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

1,4-alpha-Glucan Branching Enzyme / chemistry*
1,4-alpha-Glucan Branching Enzyme / genetics
1,4-alpha-Glucan Branching Enzyme / metabolism
Amino Acid Substitution
Carbohydrate Sequence
Chromatography, Gel
Cloning, Molecular
Enzyme Assays
Escherichia coli / genetics
Escherichia coli / metabolism
Gene Expression
Molecular Dynamics Simulation
Molecular Sequence Data
Molecular Weight
Mutagenesis, Site-Directed
Mutation*
Plant Proteins / chemistry*
Plant Proteins / genetics
Plant Proteins / metabolism
Protein Engineering
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Starch / biosynthesis*
Starch / chemistry
Structure-Activity Relationship
Zea mays / chemistry
Zea mays / enzymology*
Zea mays / genetics

Substances

Plant Proteins
Recombinant Proteins
Starch
1,4-alpha-Glucan Branching Enzyme

Grants and funding

This work was supported by the 1000-Talents Program of the Chinese Foreign Experts Bureau (www.1000plan.org) to RGG and the Australian Research Council (www.arc.gov.au) DP130102153 to AEM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.