ABSTRACT
OBJECTIVE@#To investigate the multienzyme complex formation of human malaria parasite Plasmodium falciparum (P. falciparum) orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzyme of the de novo pyrimidine biosynthetic pathway. Previously, we have clearly established that the two enzymes in the malaria parasite exist physically as a heterotetrameric (OPRT)2(OMPDC)2 complex containing two subunits each of OPRT and OMPDC, and that the complex have catalytic kinetic advantages over the monofunctional enzyme.@*METHODS@#Both enzymes were cloned and expressed as recombinant proteins. The protein-protein interaction in the enzyme complex was identified using bifunctional chemical cross-linker, liquid chromatography-mass spectrometric analysis and homology modeling.@*RESULTS@#The unique insertions of low complexity region at the α 2 and α 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-protein interaction of the heterotetrameric (OPRT)2(OMPDC)2 multienzyme 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.
Subject(s)
Humans , Amino Acid Sequence , Malaria, Falciparum , Parasitology , Models, Molecular , Molecular Sequence Data , Orotate Phosphoribosyltransferase , Chemistry , Genetics , Metabolism , Orotidine-5'-Phosphate Decarboxylase , Chemistry , Genetics , Metabolism , Plasmodium falciparum , Genetics , Protein Binding , Protein Interaction Domains and Motifs , Protozoan Proteins , Chemistry , Genetics , Metabolism , Recombinant Proteins , Chemistry , Genetics , Metabolism , Sequence AlignmentABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of orotate phosphoribosyltransferase (OPRT) in colorectal carcinoma and analyze its correlations with the toxicities of chemotherapy.</p><p><b>METHODS</b>The expression of OPRT mRNA was detected using RT-PCR in colorectal carcinoma tissues and paired adjacent normal tissues from 58 patients receiving FOLFOX6 regimen chemotherapy. The toxicities of the chemotherapy were recorded, and the correlations between OPRT mRNA expression and the toxicities were analyzed.</p><p><b>RESULTS</b>OPRT mRNA expression was significantly higher in the tumor tissues than in the corresponding normal tissues (P=0.001), but OPRT expression in the tumor tissues was not correlated with the toxicities of the chemotherapy (P>0.05). OPRT level in the normal tissues showed a significant positive correlation with the occurrence of diarrhea in these cases (P=0.013).</p><p><b>CONCLUSION</b>OPRT expression in colorectal carcinoma tissues is not correlated with the toxicities of 5-FU-based regimen, but OPRT expression in the normal tissues can help predict the toxicities associated with 5-FU.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Antimetabolites, Antineoplastic , Therapeutic Uses , Toxicity , Colorectal Neoplasms , Drug Therapy , Metabolism , Pathology , Fluorouracil , Therapeutic Uses , Toxicity , Intestinal Mucosa , Metabolism , Pathology , Orotate Phosphoribosyltransferase , MetabolismABSTRACT
Metabolic pathways in the malarial parasite are markedly different from the host, eg, hemoglobin, fatty acids, folate and nucleic acids. Understanding of metabolic function will illuminate new chemotherapeutic targets for drug development, including the identification of target(s) for drugs in current use. The parasite-contained pyrimidine biosynthetic pathway is essential for growth and development in the human host. Plasmodium falciparum carbonic anhydrase, producing HCO3- as a pyrimidine precursor, was identified as alpha- type and the encoded gene was cloned and sequenced. The first six enzymes, catalyzing the conversion of HCO3-, ATP, L-aspartate and L-glutamine to uridine 5'-monophosphate (UMP), were partially characterized. The genes encoding these enzymes were identified in order, from the first to the sixth step, as CPSII (carbamyl phosphate synthase II), ATC (aspartate transcarbamylase), DHO (dihydroorotase), DHOD (dihydroorotate dehydrogenase, DHOD), OPRT (orotate phosphoribosyltransferase, OPRT), and OMPDC (orotidine 5'-monophosphate decarboxylase, OMPDC). Unlike its analogous parasitic protozoan, Trypanosoma, the organization of the malarial genes was not an operon-like cluster. The CPSII, DHO and OPRT genes were conserved to bacterial counterparts, whereas the ATC, DHOD and OMPDC were mosaic variations. The data support the mosaic pyrimidine pathway in the malarial parasite. The human host had five enzymes out of the six associated into two different multifunctional proteins, in that a single gene CPSII-ATC-DHO encoded the first three enzymes, and another gene OPRT-OMPDC encoded the last two enzymes. In the malarial parasite, the CPSII and ATC were not characterized. The DHO was partially characterized in Plasmodium berghei. The DHOD was well characterized in both P. falciparum and P. berghei. It was functionally expressed in Escherichia coli. The physical and kinetic properties of the recombinant pfDHOD were similar to the native enzyme. The OPRT and OMPDC were also partially characterized. These lines of evidence indicate that the malarial pyrimidine enzymes are mono-functional forms. In addition, the enzymatic activities inter-converting uracil, uridine and UMP of the pyrimidine salvage pathway, were demonstrated, and the gene encoding uridine phosphorylase was cloned. Our results suggest that the pyrimidine enzymes are possible new drug targets.