3WPU

Full-length beta-fructofuranosidase from Microbacterium saccharophilum K-1

Summary for 3WPU

• Entry DOI: 10.2210/pdb3wpu/pdb
• Related: 3VSR 3VSS 3WPV 3WPY 3WPZ
• Descriptor: Beta-fructofuranosidase, GLYCEROL (3 entities in total)
• Functional Keywords: glycoside hydrolase family 68, beta-propeller, hydrolase
• Biological source: Arthrobacter
• Total number of polymer chains: 2
• Total formula weight: 119210.95

Authors

Yokoi, G.,Mori, M.,Sato, S.,Miyazaki, T.,Nishikawa, A.,Tonozuka, T. (deposition date: 2014-01-17, release date: 2014-03-12, Last modification date: 2024-10-30)

Primary citation

Ohta, Y.,Hatada, Y.,Hidaka, Y.,Shimane, Y.,Usui, K.,Ito, T.,Fujita, K.,Yokoi, G.,Mori, M.,Sato, S.,Miyazaki, T.,Nishikawa, A.,Tonozuka, T.
Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 beta-fructofuranosidase
Appl.Microbiol.Biotechnol., 98:6667-6677, 2014

PubMed Abstract: A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4(G)-β-D-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.

PubMed: 24633372
DOI: 10.1007/s00253-014-5645-3

Experimental method

X-RAY DIFFRACTION (1.6 Å)