Abstract
Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of α1,6-branching points in glycogen. Until recently it was believed that all GBEs belong to glycoside hydrolase family 13 (GH13). Here we describe the cloning and expression of the Thermus thermophilus family GH57-type GBE and report its biochemical properties and crystal structure at 1.35-Å resolution. The enzyme has a central (β/α)(7)-fold catalytic domain A with an inserted domain B between β2 and α5 and an α-helix-rich C-terminal domain, which is shown to be essential for substrate binding and catalysis. A maltotriose was modeled in the active site of the enzyme which suggests that there is insufficient space for simultaneously binding of donor and acceptor substrates, and that the donor substrate must be cleaved before acceptor substrate can bind. The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the GH13 GBEs characterized to date.
Highlights
Branching enzyme (EC 2.4.1.18; glycogen branching enzyme; GBE) catalyzes the formation of ␣1,6-branching points in glycogen
The biochemical assessment showed that the GH57 GBE possesses about 4% hydrolytic activity with amylose and in vitro forms a glucan product with a novel fine structure, demonstrating that the GH57 GBE is clearly different from the glycoside hydrolase family 13 (GH13) GBEs characterized to date
At least three enzymes are required for glycogen biosynthesis: ADP-pyrophosphorylase, which catalyzes ADP-Glc synthesis, glycogen synthase, which is involved both in initiation and elongation of the ␣1,4-linear chain [3], and glycogen branching enzyme (GBE),4 which introduces the side chains
Summary
General procedures for DNA manipulation and cloning were done as described [9]. Restriction endonucleases and T4 DNA ligase were from Fermentas GMBH and were used as recommended by the supplier. T. thermophilus HB8 was supplied by JMC RIKEN BRC (Japan). It was grown in TM medium at 70 °C and its chromosomal DNA was isolated. The gene TTHA1902 was amplified from genomic DNA by PCR using Expand High Fidelity polymerase (Fermentas) and oligonucleotides Tt-FP/Tt-RP (supplemental Table S1) and the product was cloned into pCRXL-TOPO (Invitrogen) resulting in pTt-TOPO plasmid. For expression of the protein, the gene was cloned into pET15b (Novagen) using NdeI and BglII restriction sites, leading to the pTt plasmid, which encodes TtGBE with an N-terminal His tag
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