Stabilization of Flavobacterium meningosepticum glycerol kinase by introduction of a hydrogen bond

• Published in: Bioscience, biotechnology, and biochemistry Vol. 66; no. 6; pp. 1374 - 1377
• Main Authors: Sakasegawa, S. (Asahi Chemical Industry Co. Ltd., Ohito, Shizuoka (Japan)), Takehara, H, Yoshioka, I, Misaki, H, Sakuraba, H, Ohshima, T
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.06.2002; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Amino Acid Substitution; Biological and medical sciences; Biotechnology; Enzyme Stability; FLAVOBACTERIUM; Flavobacterium - enzymology; Flavobacterium meningosepticum; Fundamental and applied biological sciences. Psychology; GLYCEROL; glycerol kinase; Glycerol Kinase - chemistry; Glycerol Kinase - genetics; Glycerol Kinase - metabolism; HEAT TOLERANCE; HYDROGEN; hydrogen bond; Hydrogen Bonding; Methods. Procedures. Technologies; Models, Molecular; Protein Conformation; Protein engineering; site-directed mutagenesis; Temperature; thermostability; TRANSFERASES; Protein engineering; Chryseobacterium meningosepticum; Flavobacteriaceae; Enzymatic activity; Enzyme; Glycerol kinase; Transferases; Bacteria; Mutation; Intramolecular hydrogen bond; Thermal stability
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.66.1374

The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp [Protein Eng., 14, 663-667 (2001)]. Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme. If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329. We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn. As we expected, S414N showed comparable thermostability to that of S329D. On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed.