Molecular cloning, expression, characterization and mutation of Plasmodium falciparum guanylate kinase.
The present work describes cloning, expression, purification, characterization, and mutation of Plasmodium falciparum guanylate kinase (PlasmoDB ID PFI1420w). Amino-acid sequence alignment revealed important differences especially in K42-V51, Y73-A77, and F100-L110, which include residues important for kinase activity, and at helix 3, which is important for domain movements. The catalytic efficiency for dGMP was 22-fold lower than that for GMP, whose value is the lowest among known guanylate kinases. dGMP was found to a competitive inhibitor for GMP with K(i)=0.148 mM and a mixed-type inhibitor with regard to ATP with measured K(i)=0.4 mM. The specificity constant (K(cat)/K(m)) of the four examined mutants varied for natural substrate GMP/dGMP, indicating the involvement of different mechanisms in substrate recognition and subsequent loop-domain movement. These results show that P. falciparum guanylate kinase is structurally and biochemically distinct from other guanylate kinases and could be a possible target in drug development.
Kandeel
Mahmoud
M
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Nakanishi
Masayuki
M
Ando
Takayuki
T
El-Shazly
Kamal
K
Yosef
Tarek
T
Ueno
Yoshihito
Y
Kitade
Yukio
Y
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D003848:Deoxyguanine Nucleotides / Q000378:metabolism / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006157:Guanosine Monophosphate / Q000378:metabolism; D051528:Guanylate Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment
D003848:Deoxyguanine Nucleotides; D004791:Enzyme Inhibitors; C007257:2'-deoxyguanosine 5'-phosphate; D006157:Guanosine Monophosphate; D000255:Adenosine Triphosphate; D051528:Guanylate Kinases