Biomaterial Database

Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues. 1996

K Watanabe, and K Kitamura, and Y Suzuki

Department of Agricultural Chemistry, Kyoto Prefectural University, Japan.

To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, and Y. Suzuki, Eur. J. Biochem. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.

UI	MeSH Term	Description	Entries
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009834	Oligo-1,6-Glucosidase	An enzyme that catalyzes the endohydrolysis of 1,6-alpha-glucosidic linkages in isomaltose and dextrins produced from starch and glycogen by ALPHA-AMYLASES. EC 3.2.1.10.	Isomaltase,Exo-Oligo-1,6-Glucosidase,Exo Oligo 1,6 Glucosidase,Oligo 1,6 Glucosidase
D011392	Proline	A non-essential amino acid that is synthesized from GLUTAMIC ACID. It is an essential component of COLLAGEN and is important for proper functioning of joints and tendons.	L-Proline,L Proline
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004269	DNA, Bacterial	Deoxyribonucleic acid that makes up the genetic material of bacteria.	Bacterial DNA
D004795	Enzyme Stability	The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat.	Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D005798	Genes, Bacterial	The functional hereditary units of BACTERIA.	Bacterial Gene,Bacterial Genes,Gene, Bacterial
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001407	Bacillus	A genus of BACILLACEAE that are spore-forming, rod-shaped cells. Most species are saprophytic soil forms with only a few species being pathogenic.	Bacillus bacterium
D001426	Bacterial Proteins	Proteins found in any species of bacterium.	Bacterial Gene Products,Bacterial Gene Proteins,Gene Products, Bacterial,Bacterial Gene Product,Bacterial Gene Protein,Bacterial Protein,Gene Product, Bacterial,Gene Protein, Bacterial,Gene Proteins, Bacterial,Protein, Bacterial,Proteins, Bacterial