Biochemical Characterization of the CDP- d -Arabinitol Biosynthetic Pathway in Streptococcus pneumoniae 17F

Abstract

Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of D-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-D-arabinitol (the nucleotide-activated form of D-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA-and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

“…Specifically, UDP/TDP-sugars (predominate in microbial natural product biosynthetic pathways) (5,20), CDP-sugars (important to the biosynthesis of pathogenic bacteria antigens) (21,22), GDP-sugars (used in a range of glycobiological processes) (5,23), and ADPsugars (important to intracellular trafficking, posttranslational modification of proteins, DNA repair, programmed cell death, and glycogen metabolism) (24)(25)(26) were targeted in this study. While UDP or TDP with 2-chloro-4-nitrophenyl glucoside were previously recognized as excellent substrates in the OleD-catalyzed sugar nucleotide syntheses reactions, ADP and GDP were identified as relatively poor substrates (i.e., seven-and 50-fold decrease in conversion for ADP and GDP, respectively, compared with equivalent reactions with UDP), while CDP was not recognized as a substrate (19).…”

Increase in prevalence of Streptococcus pneumoniae serogroup 24 in children upon introducing 13-valent pneumococcal conjugate vaccine in Japan

“…Therefore, we analysed the abpA gene sequences in serogroup 24 strains isolated in this study. The abpA gene, formerly known as abp1 , is 720 bp long and encodes a transferase enzyme (240 aa) that is involved in the biosynthesis of CDP-arabinitol [24, 25]. In our study, all 18 strains of serotype 24F ST 2572 had the same amino acid substitutions in the sequence of the protein encoded by the abpA gene.…”

Advances in enzyme substrate analysis with capillary electrophoresis

“…Boric acid is also used as a complexing agent that aids in the separation of substrates and products [36–45] based on the formation of a complex between boric acid and cis-diols. In addition to borate and highly sulfated cyclodextrins, ionic liquids are used for the separation of labelled amino acid enantiomers [46].…”

“…An off-line enzyme incubation is preferred if the working pH range of the enzyme reaction does not support the separation of product from substrate. These reactions typically use reaction volumes of 100 μL or greater [29,38,39,49,53–55], although smaller volumes have been reported [36,37]. Off-line enzyme reactions can be quenched to allow more flexibility in the separation of each sample; for example, when reactions are run in parallel.…”

“…The enzyme reactions are typically quenched by heating [38], perchloric acid [54], hydrochloric acid [39] or freezing [40] prior to analysis. Off-line kinetic studies have been performed with several enzymes, including those used in biosynthesis pathways [15,36,37] and those with enantiomers or diasteriomers as substrates [29,34,46]. …”