Biochemical Characterization and Synthetic Application of α‐1,3‐Glucosyltransferase from Pneumococcus Serotype 18C

Abstract

The enzymatic activity and function of a new α-1,3-glucosyltransferase, Cps18CU, derived from Streptococcus pneumoniae serotype 18C was identified for the first time using the enzymatically synthetic disaccharide Rhaβ1,4-Glcα-PP-O(CH 2 ) 11 -OPh as the acceptor substrate. Further investigation on substrate specificity revealed that Cps18CU exhibited broad toleration toward various NDP-Glc donors and also accepted such disaccharide acceptor containing Rhaβ1,4-Glc moiety in structure. Finally, Cps18CU was employed into a one-pot twoenzyme reaction system, furnishing trisaccharide Glcα1,3-Rhaβ1,4-Glcα-PP-O(CH 2 ) 11 -OPh in an 81 % yield.

Sequential One‐Pot Three‐Enzyme Synthesis of the Tetrasaccharide Repeating Unit of Group B Streptococcus Serotype VIII Capsular Polysaccharide

“…Excitingly, it has been disclosed that the artificially synthesized glycolipids with shorter lipid chains can act as Und surrogates that can be well recognized by various bacterial GTs. [ 26‐28 ] In our previous study, the chemically synthetic glycolipid Glcα‐PP‐ (CH 2 ) 11 ‐OPh 1 (Scheme 1), which has a 11‐phenoxyundecan‐1‐yl lipid linked to the Glc anomeric position via an α‐linked diphosphate bridge, was proved to be a recognizable acceptor substrate for β‐1,4‐rhamnosyltransferase (Cps23FT) from S. pneumonia serotype 23F. [ 27 ] Therefore, it may be used as the substrate to study the subsequent GTs.…”

Biochemical Characterization and Synthetic Application of WciN and Its Mutants From Streptococcus pneumoniae Serotype 6B

“…We have recently reported the optimized synthesis of the glycolipid Glcα-PP-(CH 2 ) 11 -OPh 1 ( Liang et al, 2022 ) and utilized it as an acceptor substrate to characterize several bacterial glycosyltransferases related to CPS RU biosynthesis ( Wang et al, 2019 ; Wang et al, 2021 ; Liang et al, 2022 ). Therefore, using it as an enzymatic substrate, the detailed biochemical properties of WciN were then investigated.…”

“…Reaction mixtures were performed for 10 min and then terminated by boiling at 100°C for 30 s. After centrifuging for 10 min under 12,000 rpm, the supernatant was analyzed with HPLC (DionexCarboPac TM PA-100 column, 4 × 250 mm, 0–1 M ammonium acetate buffer eluent). The byproduct UDP was monitored to assess the reaction process owing to its strong UV absorption at 260 nm and its convenience to be quantitated by HPLC ( Wang et al, 2021 ; Liang et al, 2022 ).…”

“…Thus, the more in vitro evidence to confirm such serotype evolution are further worthy of exploring. In this study, we presented in detail the biochemical properties of WciN derived from pneumococcus type 6B using the synthesized glycolipid Glcα-PP-(CH 2 ) 11 -OPh 1 ( Wang et al, 2019 ; Wang et al, 2021 ; Liang et al, 2022 ) ( Figure 1B ) as acceptor substrate, and carried out the single or dual amino acid substitution of D38 and/or A150 residue of WciN to verify the different glycosylation functions of the resultant glycosyltransferase mutants.…”

Producing hexamethylenediamine from caprolactam via 6-aminocapronitrile: a green production technology of the monomer of nylon-66

“…The energy change of the whole process is −10.6 kcal mol −1 , and the energy barrier of the rate-determining step is 32.2 kcal mol −1 , indicating that the Lewis acid Al 2 O 3 can effectively promote the ammoniation of CPL to AHA. Although the literature on amide ammonolysis is scarce compared to that on amide alcoholysis [33][34][35] and amide hydrolysis, [36][37][38] these reactions are all a type of nucleophilic substitution, sharing related mechanisms and catalyst systems. The use of Lewis or Brønsted acids can activate the leaving group (e.g., H 2 N-R 2 in Scheme S1 †) and lower the reaction barrier, facilitating the occurrence of reactions.…”