The hexameric
purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce
nucleoside analogues in industry and can be exploited in the development of new anti-
tumor gene
therapies. In order to provide structural basis for
enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and
nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11
ligands, including clinically relevant compounds. The crystal structure of six
ligands (
adenine, 2'
deoxyguanosine,
aciclovir,
ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxy)
ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5'
hydroxyl group of
adenosine and Arg(43*) side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and
8-bromoguanosine pointed out that the Cl(6) and Br(8) substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by
tubercidin and suggested that the acyclic
nucleoside ganciclovir is a better inhibitor for hexameric PNPs than
aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90) by a
threonine in the B. cereus hexameric
adenosine phosphorylase (Thr(91)) is responsible for the lack of negative cooperativity of
phosphate binding in this
enzyme.