PubMed · 9874653
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A K Hopper. 1998-12-11. Nuclear functions charge ahead.. https://doi.org/10.1126/science.282.5396.2003
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A simple modification procedure, the succinylation of amino groups, was suitable to increase the transferase (disproportionation) activity of cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. 501 using different linear oligosaccharides as acceptors. On the contrary, the synthesis of cyclodextrins (CDs), the coupling of CDs with oligosaccharides, and the hydrolysis of starch decreased after chemical modification. The degree of succinylation of amino groups (45%) was accurately determined by MALDI-TOF mass spectrometry. The formation of CDs under industrial conditions was analyzed for native and succinylated CGTases, showing similar selectivity to alpha-, beta-, gamma-CD. The acceptor reaction with D-glucose using soluble starch as glucosyl donor was studied at 60 degrees C and pH 5.5. Malto-oligosaccharides (MOS) production was notably higher using the semisynthetic enzyme at different ratios (w/w) starch:D-glucose. Thus, more than 90% of the initial starch was converted into MOS (G2-G7) in 48 h employing a ratio donor:acceptor 1:2 (w/w).
Esters were prepared by acylation of hydroxypropyl cellulose with fatty acid catalyzed by immobilized lipase from Candida antarctica in tert-butanol. The nature of the substrates used, the initial water activity of the system, and the molecular weight of the hydroxypropyl cellulose were investigated. Moreover, Fourier transform-infrared (FT-IR) spectroscopy was used for determination of ester content on hydroxypropyl cellulose. Specifically, a linear relationship was established between the peak height assigned to the absorption of the esterified carboxyl groups of the cellulose and the ester content. At optimum reaction conditions, the ester content on the hydroxypropyl cellulose was about 11%.
We have investigated the role of 2'-OH groups in the specific interaction between the acceptor stem of Escherichia coli tRNA(Cys) and cysteine-tRNA synthetase. This interaction provides for the high aminoacylation specificity observed for cysteine-tRNA synthetase. A synthetic RNA microhelix that recapitulates the sequence of the acceptor stem was used as a substrate and variants containing systematic replacement of the 2'-OH by 2'-deoxy or 2'-O-methyl groups were tested. Except for position U73, all substitutions had little effect on aminoacylation. Interestingly, the deoxy substitution at position U73 had no effect on aminoacylation, but the 2'-O-methyl substitution decreased aminoacylation by 10-fold and addition of the even bulkier 2'-O-propyl group decreased aminoacylation by another 2-fold. The lack of an effect by the deoxy substitution suggests that the hydrogen bonding potential of the 2'-OH at position U73 is unimportant for aminoacylation. The decrease in activity upon alkyl substitution suggests that the 2'-OH group instead provides a monitor of the steric environment during the RNA-synthetase interaction. The steric role was confirmed in the context of a reconstituted tRNA and is consistent with the observation that the U73 base is the single most important determinant for aminoacylation and therefore is a site that is likely to be in close contact with cysteine-tRNA synthetase. A steric role is supported by an NMR-based structural model of the acceptor stem, together with biochemical studies of a closely related microhelix. This role suggests that the U73 binding site for cysteine-tRNA synthetase is sterically optimized to accommodate a 2'-OH group in the backbone, but that the hydroxyl group itself is not involved in specific hydrogen bonding interactions.