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

Abstract

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 d -xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to d -xylulose-5-phosphate ( d -Xlu-5-P) to form CDP- d -xylulose, and abpB was characterized to be a CDP- d -xylulose reductase-encoding gene, responsible for the conversion of CDP- d -xylulose to CDP- d -arabinitol as the final product. The kinetic parameters of AbpA for the substrates d -Xlu-5-P and CTP and those of AbpB for the substrate CDP- d -xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP- d -arabinitol.