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Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase.
Parasitol Int. 2018 Feb; 67(1):79-84.PI

Abstract

Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having a COOH-terminal orotate phosphoribosyltransferase (OPRT) and an NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, were stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (kcat/Km). The kcat was selectively enhanced up to three orders of magnitude, while the Km was not much affected and remained at low μM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a "super-enzyme" with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivities and metabolic functions on molecular evolution.

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Authors+Show Affiliations

Paojinda P
Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand; Inter-Department Program of Biomedical Science, Faculty of Graduate School, Chulalongkorn University, Bangkok 10330, Thailand.
Imprasittichai W
Department of Basic Medical Science, Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok 10300, Thailand.
Krungkrai SR
Unit of Biochemistry, Department of Medical Science, Faculty of Science, Rangsit University, Patumthani 12000, Thailand.
Palacpac NMQ
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Horii T
Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Krungkrai J
Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: jerapan.k@chula.ac.th.

MeSH

Artificial Gene FusionEscherichia coliGene FusionMicroorganisms, Genetically-ModifiedOrotate PhosphoribosyltransferaseOrotidine-5'-Phosphate DecarboxylasePlasmodium falciparumProtozoan Proteins

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28389349

Citation

Paojinda, Patsarawadee, et al. "Bifunctional Activity of Fused Plasmodium Falciparum Orotate Phosphoribosyltransferase and Orotidine 5'-monophosphate Decarboxylase." Parasitology International, vol. 67, no. 1, 2018, pp. 79-84.
Paojinda P, Imprasittichai W, Krungkrai SR, et al. Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase. Parasitol Int. 2018;67(1):79-84.
Paojinda, P., Imprasittichai, W., Krungkrai, S. R., Palacpac, N. M. Q., Horii, T., & Krungkrai, J. (2018). Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase. Parasitology International, 67(1), 79-84. https://doi.org/10.1016/j.parint.2017.04.003
Paojinda P, et al. Bifunctional Activity of Fused Plasmodium Falciparum Orotate Phosphoribosyltransferase and Orotidine 5'-monophosphate Decarboxylase. Parasitol Int. 2018;67(1):79-84. PubMed PMID: 28389349.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Bifunctional activity of fused Plasmodium falciparum orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase. AU - Paojinda,Patsarawadee, AU - Imprasittichai,Waranya, AU - Krungkrai,Sudaratana R, AU - Palacpac,Nirianne Marie Q, AU - Horii,Toshihiro, AU - Krungkrai,Jerapan, Y1 - 2017/04/04/ PY - 2017/03/13/received PY - 2017/03/31/revised PY - 2017/04/03/accepted PY - 2017/4/9/pubmed PY - 2018/6/16/medline PY - 2017/4/9/entrez KW - Bifunctional fused enzyme KW - Catalytic efficiency KW - Orotate phosphoribosyltransferase KW - Orotidine 5′-monophosphate decarboxylase KW - Plasmodium falciparum SP - 79 EP - 84 JF - Parasitology international JO - Parasitol Int VL - 67 IS - 1 N2 - Fusion of the last two enzymes in the pyrimidine biosynthetic pathway in the inversed order by having a COOH-terminal orotate phosphoribosyltransferase (OPRT) and an NH2-terminal orotidine 5'-monophosphate decarboxylase (OMPDC), as OMPDC-OPRT, are described in many organisms. Here, we produced gene fusions of Plasmodium falciparum OMPDC-OPRT and expressed the bifunctional protein in Escherichia coli. The enzyme was purified to homogeneity using affinity and anion-exchange chromatography, exhibited enzymatic activities and functioned as a dimer. The activities, although unstable, were stabilized by its substrate and product during purification and long-term storage. Furthermore, the enzyme expressed a perfect catalytic efficiency (kcat/Km). The kcat was selectively enhanced up to three orders of magnitude, while the Km was not much affected and remained at low μM levels when compared to the monofunctional enzymes. The fusion of the two enzymes, creating a "super-enzyme" with perfect catalytic power and more flexibility, reflects cryptic relationship of enzymatic reactivities and metabolic functions on molecular evolution. SN - 1873-0329 UR - https://www.unboundmedicine.com/medline/citation/28389349/Bifunctional_activity_of_fused_Plasmodium_falciparum_orotate_phosphoribosyltransferase_and_orotidine_5'_monophosphate_decarboxylase_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1383-5769(17)30126-5 DB - PRIME DP - Unbound Medicine ER -

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