A unique insertion of low complexity amino acid sequence underlies protein-protein interaction in human malaria parasite orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase

Objective:To investigate the multienzyine complex formation of human malaria parasite Plasmodium falciparum[P.falciparum)orotate phosphoribosyltransferase(OPRT)and orotidine5'-monophosphate decarboxylase(OMPDC),the fifth and sixth enzyme of the de novo pyrimidine biosynthetic palhway.Previously,we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric(OPRT)_2(OMPDG)_2 complex containing two subunits each of OPRT and OMPDC.and that the complex have catalytic kinetic advantages over the monofunetional enzyme.Methods:Both enzymes were cloned and expressed as recombinant proteins.The protein-protein interaction in the enzyme complex was identified using bifunctionul chemical cross-linker,liquid chromatography-mass spectrometric analysis and homology modeling,Results:The unique insertions of low complexity region at the a 2 and a 5 helices of the parasite OMPDC,characterized by single amino acid repeat sequence which was not found in homologous proteins from other organisms,was located on the OPRT-OMPDC interface.The structural models for the protein-prolein interaction of the helerotetrameric(OPRT)_2(OMPDC)_2multienzyme complex were proposed.Conclusions:Based on the proteomic data and structural modeling,it is surmised that the human malaria parasite low complexity region is responsible for the OPRT-OMPDC interaction.The structural complex of the parasite enzymes,thus,represents an efficient functional kinetic advantage,which in line with co-localization principles of evolutional origin,and allosteric control in protein-protein-interactions.

Orotidine Monophosphate Decarboxylase——A Fascinating Workhorse Enzyme with Therapeutic Potential

Orotidine 5＇-monophosphate decarboxylase （ODCase） is known as one of the most proficient enzymes. The enzyme catalyzes the last reaction step of the de novo pyrimidine biosynthesis, the conversion from orotidine 5＇-monophosphate （OMP） to uridine 5＇-mono- phosphate. The enzyme is found in all three domains of life, Bacteria, Eukarya and Archaea. Multiple sequence alignment of 750 putative ODCase sequences resulted in five distinct groups. While the universally conserved DxKxxDx motif is present in all the groups, depending on the groups, several characteristic motifs and residues can be identified. Over 200 crystal structures of ODCases have been determined so far. The structures, together with biochemical assays and computational studies, elucidated that ODCase utilized both transition state stabilization and substrate distortion to accelerate the decarboxylation of its natural substrate. Stabilization of the vinyl anion intermediate by a conserved lysine residue at the catalytic site is considered the largest contributing factor to catalysis, while bending of the carboxyl group from the plane of the aromatic pyrimidine ring of OMP accounts for substrate distortion. A number of crystal structures of ODCases complexed with potential drug candidate molecules have also been determined, including with 6-iodo- uridine, a potential antimalarial agent.

pyrG is required for maintaining stable cellular uracil level and normal sporulation pattern under excess uracil stress in Aspergillus nidulans

Tight control of the intracellular uracil level is believed to be important to reduce the occurrence of uracil incorporation into DNA. The pyrG gene ofAspergillus niduIans encodes orotidine 5＇-phosphate decarboxylase, which catalyzes the conversion of orotidine monophosphate （OMP） to uridine monophosphate （UMP）. In this study, we found that pyrG is critical for maintaining uracil at a low concentration in A. nidulans cells in the presence of exogenous uracil. Excess uracil and its derivatives had a stronger inhibitory effect on the growth of the pyrG89 mutant with defective OMP decarboxylase activity than on the growth of wild type, and induced sexual development in the pyrG89 mutant but not in wild type. Analysis of transcriptomic responses to excess uracil by digital gene expression profiling （DGE） revealed that genes related to sexual development were transcrip- tionally activated in the pyrG89 mutant but not in wild type. Quantitative analysis by HPLC showed that the cellular uracil level was 6.5 times higher in the pyrG89 mutant than in wild type in the presence of exogenous uracil. This study not only provides new information on uracil recycling and adaptation to excess uracil but also reveals the potential effects of OMP decarboxylase on fungal growth and development.