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Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach.

ABSTRACT: A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7-14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr(451)-Val(1082)), a portion of which shares identity (35% at Ala(39)-Ser(1304) of PsDex) with Pro(32)-Ala(755) of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val(837)-Met(932) for PsDex and Tyr(404)-Tyr(492) for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala(39)-Ser(1304)) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp(189), Asp(340), Glu(412), and Asp(1254) of PsDex) of catalytic candidates. Their amide mutants decreased activity (1?1,500 to 1?40,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using ?-isomaltotetraosyl fluoride with NaN(3). D340G or E412Q formed a ?- or ?-isomaltotetraosyl azide, respectively, strongly indicating Asp(340) and Glu(412) as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from ?-isomaltotetraosyl fluoride in the presence of NaN(3).

SUBMITTER: Kim YM

PROVIDER: S-EPMC3370177 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach.

Kim Young-Min YM Kiso Yoshiaki Y Muraki Tomoe T Kang Min-Sun MS Nakai Hiroyuki H Saburi Wataru W Lang Weeranuch W Kang Hee-Kwon HK Okuyama Masayuki M Mori Haruhide H Suzuki Ryuichiro R Funane Kazumi K Suzuki Nobuhiro N Momma Mitsuru M Fujimoto Zui Z Oguma Tetsuya T Kobayashi Mikihiko M Kim Doman D Kimura Atsuo A

The Journal of biological chemistry 20120329 24

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7-14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr(451)-Val(1082)), a portion of which shares identity (35% at Ala(39)-Ser(1304) of PsDex) with Pro(32)-Ala(755) of CI glucanotransferase ( ...[more]

PMID: 22461618

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Project description:Alpha-amino acids are the building blocks of proteins and are widely used as components of medicinally active molecules and chiral catalysts. Efficient chemo-enzymatic methods for the synthesis of enantioenriched alpha-amino acids have been developed, but it is still a challenge to obtain non-natural amino acids. Alkene hydrogenation is broadly useful for the enantioselective catalytic synthesis of many classes of amino acids, but it is not possible to obtain alpha-amino acids bearing aryl or quaternary alkyl alpha-substituents using this method. The Strecker synthesis-the reaction of an imine or imine equivalent with hydrogen cyanide, followed by nitrile hydrolysis-is an especially versatile chemical method for the synthesis of racemic alpha-amino acids. Asymmetric Strecker syntheses using stoichiometric amounts of a chiral reagent have been applied successfully on gram-to-kilogram scales, yielding enantiomerically enriched alpha-amino acids. In principle, Strecker syntheses employing sub-stoichiometric quantities of a chiral reagent could provide a practical alternative to these approaches, but the reported catalytic asymmetric methods have seen limited use on preparative scales (more than a gram). The limited utility of existing catalytic methods may be due to several important factors, including the relatively complex and precious nature of the catalysts and the requisite use of hazardous cyanide sources. Here we report a new catalytic asymmetric method for the syntheses of highly enantiomerically enriched non-natural amino acids using a simple chiral amido-thiourea catalyst to control the key hydrocyanation step. This catalyst is robust, without sensitive functional groups, so it is compatible with aqueous cyanide salts, which are safer and easier to handle than other cyanide sources; this makes the method adaptable to large-scale synthesis. We have used this new method to obtain enantiopure amino acids that are not readily prepared by enzymatic methods or by chemical hydrogenation.

Dataset Information

Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach.

Publications

Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach.

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