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Effect of Leu²⁷⁷ on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin Glucosyltransferase.

ABSTRACT: The disproportionation activity of cyclodextrin glucosyltransferase (CGTase; EC 2.4.1.19) can be used to convert small molecules into glycosides, thereby enhancing their solubility and stability. However, CGTases also exhibit a competing hydrolysis activity. The +2 subsite of the substrate binding cleft plays an important role in both the disproportionation and hydrolysis activities, but almost all known mutations at this site decrease disproportionation activity. In this study, Leu²⁷⁷ of the CGTase from Bacillus stearothermophilus NO2, located near both the +2 subsite and the catalytic acid/base Glu²⁵³, was modified to assess the effect of side chain size at this position on disproportionation and hydrolysis activities. The best mutant, L277M, exhibited a reduced K_m for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased k_cat/K_m (1,175 s^-1 mM^-1 versus 686.1 s^-1 mM^-1), compared with those of the wild-type enzyme. The disproportionation-to-hydrolysis ratio of L277M was 2.4-fold greater than that of the wild type. Existing structural data were combined with a multiple-sequence alignment and Gly²⁸² mutations to examine the mechanism behind the effects of the Leu²⁷⁷mutations. The Gly²⁸² mutations were included to aid a molecular dynamics (MD) analysis and the comparison of crystal structures. They reveal that changes to a hydrophobic cluster near Glu²⁵³ and the hydrophobicity of the +2 subsite combine to produce the observed effects. IMPORTANCE In this study, mutations that enhance the disproportionation to hydrolysis ratio of a CGTase have been discovered. For example, the disproportionation-to-hydrolysis ratio of the L277M mutant of Bacillus stearothermophilus NO2 CGTase was 2.4-fold greater than that of the wild type. The mechanism behind the effects of these mutations is explained. This paper opens up other avenues for future research into the disproportionation and hydrolysis activities of CGTases. Productive mutations are no longer limited to the acceptor subsite, since mutations that indirectly affect the acceptor subsite also enhance enzymatic activity.

SUBMITTER: Kong D

PROVIDER: S-EPMC8174761 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:1. Particulate enzyme preparations obtained from Bacillus stearothermophilus B65 by digestion with lysozyme were shown to catalyse teichoic acid synthesis. With CDP-glycerol as sole substrate the preparations synthesized 1,3-poly(glycerol phosphate). It was characterized by alkaline hydrolysis, by glucosylation to the alkali-stable 2-glucosyl-1,3-poly(glycerol phosphate) with excess of UDP-glucose and a Bacillus subtilis Marburg enzyme system, by degradation of this latter product with 60%HF and periodate oxidation of the resulting glucosylglycerol. The specificity of the B. subtilis system previously reported (Glaser & Burger, 1964), was confirmed in the present work. 2. Pulse-labelling experiments, followed by periodate oxidation of the product and isolation of formaldehyde from the glycerol terminus of the polymer, showed that the B. stearothermophilus enzyme system transferred glycerol phosphate units to the glycerol end of the chain. The transfer reaction was irreversible. It was not determined if these poly(glycerol phosphate) chains were synthesized de novo, but it was shown that the newly synthesized oligomers were bound to much larger molecules. 3. When the B. stearothermophilus enzyme system was supplied with both CDP-glycerol and UDP-glucose, 1-glucosyl-2,3-poly(glycerol phosphate) was synthesized in addition to the 1,3-isomer. The former polymer was characterized by acid and alkaline hydrolysis, degradation with HF and periodate oxidation of the resulting glucosylglycerol, and periodate oxidation of the intact polymer followed by mild acid hydrolysis. This latter procedure removed the glucose substituents without disrupting the poly(glycerol phosphate) chain. 4. The poly(glycerol phosphate) isomers were distinguished by glucosylation with the B. subtilis enzymes and alkaline hydrolysis, the 2,3-isomer remaining alkali-labile. The proportion of 2,3-poly(glycerol phosphate) in the product increased with increasing amounts of UDP-glucose in the incubation mixture, but the total glycerol phosphate incorporated into products remained constant. It is suggested that the synthetic pathways of the two poly(glycerol phosphate) species may share a rate-limiting step.

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Effect of Leu²⁷⁷ on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin Glucosyltransferase.

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Effect of Leu277 on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin Glucosyltransferase.

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Effect of Leu²⁷⁷ on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin Glucosyltransferase.