8XXA

Rhodothermus marinus alpha-amylase RmGH13_47A CBM48-A-B-C domains in complex with branched pentasaccharide

Summary for 8XXA

• Entry DOI: 10.2210/pdb8xxa/pdb
• Related PRD ID: PRD_900009
• Descriptor: alpha-amylase, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-6)-[alpha-D-glucopyranose-(1-4)]alpha-D-glucopyranose-(1-4)-beta-D-glucopyranose, alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose-(1-4)-alpha-D-glucopyranose, ... (10 entities in total)
• Functional Keywords: alpha-amylase, (beta/alpha)8 barrel, carbohydrate, hydrolase
• Biological source: Rhodothermus marinus JCM 9785
• Total number of polymer chains: 1
• Total formula weight: 77507.52

Authors

Tonozuka, T. (deposition date: 2024-01-18, release date: 2024-02-07, Last modification date: 2024-11-13)

Primary citation

Miyasaka, Y.,Yokoyama, K.,Kozono, T.,Kitano, Y.,Miyazaki, T.,Sakaguchi, M.,Nishikawa, A.,Tonozuka, T.
Structural basis for the recognition of alpha-1,6-branched alpha-glucan by GH13_47 alpha-amylase from Rhodothermus marinus.
Proteins, 92:984-997, 2024

PubMed Abstract: Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides.

PubMed: 38641972
DOI: 10.1002/prot.26695

Experimental method

X-RAY DIFFRACTION (1.55 Å)