Comparison of Chain-Length Preferences and Glucan Specificities of Isoamylase-Type α-Glucan Debranching Enzymes from Rice, Cyanobacteria, and Bacteria.

Taiki Kobayashi,Satoshi Sasaki,Naoko Fujita,Takayuki Sawada,Steven Ball,Akiko Kubo,Yasunori Nakamura,Yoshinori Utsumi,Kazuhiro Umeda,Jun-Ichi Abe,Christophe Colleoni

doi:10.1371/journal.pone.0157020

Abstract

It has been believed that isoamylase (ISA)-type α-glucan debranching enzymes (DBEs) play crucial roles not only in α-glucan degradation but also in the biosynthesis by affecting the structure of glucans, although molecular basis on distinct roles of the individual DBEs has not fully understood. In an attempt to relate the roles of DBEs to their chain-length specificities, we analyzed the chain-length distribution of DBE enzymatic reaction products by using purified DBEs from various sources including rice, cyanobacteria, and bacteria. When DBEs were incubated with phytoglycogen, their chain-length specificities were divided into three groups. First, rice endosperm ISA3 (OsISA3) and Eschericia coli GlgX (EcoGlgX) almost exclusively debranched chains having degree of polymerization (DP) of 3 and 4. Second, OsISA1, Pseudomonas amyloderamosa ISA (PsaISA), and rice pullulanase (OsPUL) could debranch a wide range of chains of DP≧3. Third, both cyanobacteria ISAs, Cyanothece ATCC 51142 ISA (CytISA) and Synechococcus elongatus PCC7942 ISA (ScoISA), showed the intermediate chain-length preference, because they removed chains of mainly DP3-4 and DP3-6, respectively, while they could also react to chains of DP5-10 and 7–13 to some extent, respectively. In contrast, all these ISAs were reactive to various chains when incubated with amylopectin. In addition to a great variation in chain-length preferences among various ISAs, their activities greatly differed depending on a variety of glucans. Most strikingly, cyannobacteria ISAs could attack branch points of pullulan to a lesser extent although no such activity was found in OsISA1, OsISA3, EcoGlgX, and PsaISA. Thus, the present study shows the high possibility that varied chain-length specificities of ISA-type DBEs among sources and isozymes are responsible for their distinct functions in glucan metabolism.

Highlights

Introduction αGlucan debranching enzymes (DBE) cleave the α-1,6 glucosidic linkages of branched α-glucans such as amylopectin and glycogen
The amounts of liberated chains from the substrate were measured by the bicinchonic acid (BCA) method and the chain-length distribution of the liberated chains was analyzed by the fluorophore-assisted carbohydrate electrophoresis (FACE) method to examine the chain-length preferences of DBEs
The present study revealed a great variation in enzymatic properties of ISA-type DBEs among plants, cyanobacteria, and bacteria

Summary

Introduction

Introduction αGlucan debranching enzymes (DBE) cleave the α-1,6 glucosidic linkages of branched α-glucans such as amylopectin and glycogen. It is generally accepted that plant DBEs play important roles in a complete starch degradation by cleaving the α-1,6 glucosidic linkages because other enzymes involved in starch breakdown such as α-amylase, β-amylase, plastidial α-glucan phosphorylase (Pho1), and plastidial disproportionating enzyme (DPE1) are capable of hydrolysis and/or transfer of the α-1,4 glucosidic linkages, but not the α-1,6 glucosidic linkages. ISA and PUL have distinct glucan specificities. Past biochemical and molecular studies supported that ISA1 plays an essential role in starch biosynthesis and PUL supports the role of ISA1 to some extent at least in some cereal endosperm, whereas ISA3 is involved in catabolism of starch in plant tissues [35]

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Conclusion