Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

Abstract

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 Arg43* 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 Cl6 and Br8 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 Ser90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr91) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.

Can Crystal Symmetry and Packing Influence the Active Site Conformation of Homohexameric Purine Nucleoside Phosphorylases?

“…The script uses excellent cctbx library [16] to process a PDB file, and automatically identify whether monomers are in open or closed active site conformation. Due to the difference in numbering and different chain lengths in different PNPs, it was not possible to automate the process of detecting the open and closed conformations by for instance measuring some distances on certain amino acid numbers.…”

Crystal structure ofEscherichia colipurine nucleoside phosphorylase complexed with acyclovir

“…The structure of the homologous hexameric PNP from B. subtilis complexed with ACV has been determined at 2 Å resolution (de Giuseppe et al, 2012). The degree of sequence homology between the B. subtilis and E. coli PNPs (56% identity) is rather high.…”

“…It has a moderate inhibitory effect on human PNP (Tuttle & Krenitsky, 1984). The structure of the complex with ACV is known for human PNP (dos Santos et al, 2003), Mycobacterium tuberculosis PNP (Caceres et al, 2012) and B. subtilis PNP (de Giuseppe et al, 2012). Here, we present the three-dimensional structure of the E. coli PNP-ACV complex and compare this structure with the previously determined structures of PNP complexes.…”

Strained Conformations of Nucleosides in Active Sites of Nucleoside Phosphorylases

“…It is worth noting that not all atoms of the nucleoside are well fitted to the 2F0-Fc electron density at the 1σ level. Hence, the conclusions of the study [47] should be considered with caution.…”

“…It is worth noting that not all atoms of the nucleoside are well fitted to the 2F 0 -Fc electron density at the 1σ level. Hence, the conclusions of the study [47] should be considered with caution. Table 2 presents the conformational parameters of nucleosides in complexes with trimeric Mycobacterium tuberculosis, Schistosoma mansoni, and calf spleen PNPs, and in the complex with trimeric MTAP from human placenta.…”