REASSESSMENT OF THE CATALYTIC ACTIVITY AND SUBSTRATE SPECIFICITY OF FKBP35 FROM Plasmodium knowlesi USING PROTEASE-FREE ASSAY
DOI:
https://doi.org/10.51200/bijb.v1i.2602Keywords:
malaria, Plasmodium knowlesi, Peptidyl prolyl cis-trans isomerases, FKBPAbstract
FK506-binding protein35 of Plasmodium knowlesi (Pk-FKBP35) is a member of peptidyl prolyl cis-trans isomerase (PPIase) and is considered as a promising avenue of antimalarial drug target development. This protein is organized into the N-terminal domain responsible for PPIase catalytic activity followed and the tetratricopeptide repeat domain for its dimerization. The protease-coupling and protease-free assays are known to be the common methods for investigating the catalytic properties of PPIase. Earlier, the protease-coupling assay was used to confirm the catalytic activity of Pk-FKBP35 in accelerating cis-trans isomerization of the peptide substrate. This report is aimed to re-assess the catalytic and substrate specificity of Pk-FKBP35 using an alternative method of a protease-free assay. The result indicated that while Pk-FKBP35 theoretically contained many possible cleavage sites of chymotrypsin, experimentally, the catalytic domain was relatively stable from chymotrypsin. Furthermore, under protease-free assay, Pk-FKBP35 also demonstrated remarkable PPIase catalytic activity with kcat/KM of 4.5 + 0.13 × 105 M−1 s−1, while the kcat/KM of active site mutant of D55A is 0.81 + 0.05 × 105 M−1 s−1. These values were considered comparable to kcat/KM obtained from the protease-coupling assay. Interestingly, the substrate specificities of Pk-FKBP35 obtained from both methods are also similar, with the preference of Pk-FKBP35 towards Xaa at P1 position was Leu>Phe>Lys>Trp>Val>Ile>His>Asp>Ala>Gln>Glu. Altogether, we proposed that protease-free and protease-coupling assays arereliable for Pk-FKBP35.
References
Ahmad, S., Kumar, V., Ramanand, B., & Rao, N. M. (2011). Probing protein stability and proteolytic
resistance by loop scanning: A comprehensive mutational analysis. Protein Science, 21,
– 446. DOI: 10.1002/pro.2029
Alag, R., Bharatham, N., Dong, A., Hills, T., Harikishore, A., Widjaja, A.A, … Yoon, H. S, (2009).
Crystallographic structure of the tetratricopeptide repeat domain of Plasmodium
falciparum FKBP35 and its molecular interaction with Hsp90 C-terminal pentapeptide.
Protein Science: a Publication of the Protein Society, 18 (10), 2115 – 2124. DOI: 10.1002/
pro.226.
Berger, A., & Schechter, I. (1970). Mapping the active site of papain with the aid of peptide
substrates and inhibitors. Philosophical transactions of the Royal Society of London.
Series B, Biological Sciences, 257 (813), 249 – 264. DOI: 10.1098/rstb.1970.0024
Bisswanger, H. (2014). Enzyme assays. Perspectives in Science, 1, 41 – 55. DOI: http://dx.doi.
org/10.1016/j.pisc.2014.02.005
Budiman, C., Bando, K., Angkawidjaja, C., Koga, Y., Takano, K., & Kanaya, S. (2009). Engineering
of monomeric FK506-binding protein 22 with peptidyl prolyl cis-trans isomerase. FEBS
Journal, 276 (15), 4091 – 4101. DOI: https://doi.org/10.1111/j.1742-4658.2009.07116.x
Budiman, C., Lindang, H. U., Cheong, B. E., & Rodrigues, K. F. (2018). Inhibition and substrate
specificity properties of FKBP22 from a psychrotrophic bacterium, Shewanella sp. SIB1.
Protein Journal, 37 (3), 270 – 279. DOI: https://doi.org/10.1007/s10930-018-9772-z
Budiman, C., Tadokoro, T., Angkawidjaja, C., Koga, Y., & Kanaya, S. (2012). Role of polar and
nonpolar residues at the active site for PPIase activity of FKBP22 from Shewanella sp.
SIB1. FEBS Journal, 279 (6), 976 – 986. DOI: 10.1111/j.1742-4658.2012.08483.x
Divis, P. C., Hu, T. H., Kadir, K. A., Mohammad, D., Hii, K.C., Daneshvar, C., … Singh, B. (2020).
Efficient surveillance of Plasmodium knowlesi genetic subpopulations, Malaysian
Borneo, 2000–2018. Emerging Infectious Diseases, 26 (7), 1392 – 1398. DOI: https://
dx.doi.org/10.3201/eid2607.190924
Fanghanel, J., & Fischer, G. (2004). Insight into catalytic mechanism of peptidyl prolyl cis/trans
isomerase. Frontiers Bioscience, 9, 3453 – 3478. DOI: 10.2741/1494.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R. D., & Bairoch, A. (2005).
Protein identification and analysis tools on the ExPASy server. In J. M. Walker (Ed.), The
proteomics protocols handbook (pp. 571 – 607). Totowa, New Jersey: Humana Press.
Goh, C. K. W., Silvester, J., Wan Mahadi, W. N. S., Lee, P. C., Lau, T. Y., Thean, C. L., … Budiman,
C. (2018). Expression and characterization of functional domains of FK506-binding
protein 35 from Plasmodium knowlesi. Protein Engineering, Design and Selection, 31
(12), 489 – 498. DOI: 10.1093/protein/gzz008
Goh, X. T., Lim, Y. A., Vythilingam, I., Chew, C. H., Lee, P. C., Tan, T. C., ... Chua, K. H. (2013).
Increased detection of Plasmodium knowlesi in Sandakan division, Sabah as revealed
by PlasmoNex™. Malaria Journal, 12, 264. DOI: https://doi.org/10.1186/1475-2875-
-264
Goodwin, T. W., & Morton, R. A. (1946) The spectrophotometric determination of tyrosine
and tryptophan in proteins. Biochemical Journal, 40 (5 – 6), 628 – 632. DOI: 10.1042/
bj0400628
Harikishore, A., Niang, M., Rajan, S., Preiser, P. R., & Yoon, H. S. (2013). Small molecule Plasmodium
FKBP35 inhibitor as a potential antimalaria agent. Scientific Reports, 3, 2501. DOI:
https://doi.org/10.1038/srep02501
Ikura, T., & Ito, N. (2007). Requirements for peptidyl-prolyl isomerization activity: a
comprehensive mutational analysis of the substrate-binding cavity of FK506-binding
protein 12. Protein Science, 16 (12), 2618 – 2625. DOI: 10.1110/ps.073203707
Inoue, Y., Ogawa, Y., Kinoshita, M., Terahara, N., Shimada, M., Kodera, N., … Minamino, T. (2019).
Structural insights into the substrate specificity switch mechanism of the type III
protein export apparatus. Structure, 27 (6), 965 – 976. DOI: 10.1016/j.str.2019.03.017
Janowski, B., Wöllner, S., Schutkowski, M., & Fischer, G. (1997). A protease-free assay for
peptidyl prolyl cis/trans isomerases using standard peptide substrates. Analytical
Biochememistry, 252 (2), 299 – 307. DOI: 10.1006/abio.1997.2330
Kyte, J., & Doolittle, R. F. (1983). A simple method for displaying the hydropathic character of a
protein. Journal of Molecular Biology, 157 (1), 105 – 132.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage. Nature, 227, 680 – 685.
Lau, T. Y., Joveen-Neoh, W. F., & Chong, K. L. (2011). High Incidence of Plasmodium knowlesi
Infection in the Interior Division of Sabah, Malaysian Borneo. International Journal of
Bioscience, Biochemistry and Bioinformatics, 1 (2), 163 – 167. DOI: 10.7763/IJBBB.2011.
V1.30
Kofron, J. L., Kuzmic, P., Kishore, V., Gemmecker, G., Fesik, S. W., & Rich, D. H. (1992). Lithium
chloride perturbation of cis-trans peptide bond equilibria: effect on conformational
equilibria in cyclosporin A and on time-dependent inhibition of cyclophilin. Journal
of the American Chemical Society, 114 (7), 2670 – 2675. DOI: https://doi.org/10.1021/
ja00033a047
Parker, J. L., Corey, R. A., Stansfeld, P. J., & Newstead, S. (2019). Structural basis for substrate
specificity and regulation of nucleotide sugar transporters in the lipid bilayer. Nature
Communications, 10, 4657. DOI: https://doi.org/10.1038/s41467-019-12673-w
Parsell, D. A., & Sauer, R. T. (1989). The structural stability of a protein is an important determinant
of its proteolytic susceptibility in Escherichia coli. The Journal of Biological Chemistry,
(13), 7590 – 7595.
Rahfeld, J. U., Rucknagel, K. P., Schelbert, B., Ludwig, B., Hacker, J., Mann, K., & Fischer, G. (1994).
Confirmation of the existence of a third family among peptidyl-prolyl cis/trans
isomerases. Amino acid sequence and recombinant production of parvulin. FEBS
Letters, 352 (2), 180 – 184. DOI: https://doi.org/10.1016/0014-5793(94)00932-5
Rahfeld, J. U., Rucknagel, K. P., Stoller, G., Horne, S. M., Schierhorn, A., Young, K. D., & Fischer, G.
(1996). Isolation and amino acid sequence of a new 22-kDa FKBP-like peptidyl-prolyl
cis/trans-isomerase of Escherichia coli similarity to Mip-like proteins of pathogenic
bacteria. The Journal of Biological Chemistry, 271 (36), 22130 – 22138.
Rajharam, G. S., Cooper, D. J., William, T., Grigg, M. J., Anstey, N. M., & Barber, B. E. (2019).
Deaths from Plasmodium knowlesi Malaria: Case series and systematic review. Clinical
Infectious Diseases, 69 (10), 1703 – 1711. DOI: 10.1093/cid/ciz011
Schiene, C., Reimer, U., Schutkowski, M., & Fischer, G. (1998). Mapping the stereospecificity
of peptidyl prolyl cis/trans isomerases. FEBS Letter, 432 (3), 202 – 206. DOI: 10.1016/
s0014-5793(98)00871-0
Schiene-fischer, C., Habazetti, J., Tradler, T., & Fischer, G. (2002). Evaluation of similarities in the
cis/trans isomerase function of trigger factor and DnaK. Biological Chemistry, 383, 1865
– 1873. DOI: 10.1515/BC.2002.210
Silvester, J., Lindang, H. U., Chin, L. P., Ying, L. T., & Budiman, C. (2017). Structure and molecular
dynamic regulation of FKBP35 from Plasmodium knowlesi by structural homology
modeling and electron microscopy. Journal of Biological Sciences, 17 (8), 369 – 380.
DOI: 10.3923/jbs.2017.369.380
Singh, B., & Daneshvar, C. (2013). Human infections and detection of Plasmodium knowlesi.
Clinical Microbiology Reviews, 26 (2), 165 – 184. DOI: 10.1128/CMR.00079-12
Singh, B., Sung, L. K., Matusop, A., Radhakrishnan, A., Shamsul, S. S. G., Cox-Singh, J., ... Conway,
D. J. (2004). A large focus of naturally acquired Plasmodium knowlesi infections in
human beings. Lancet, 363 (9414), 1017 – 1024. DOI: 10.1016/S0140-6736(04)15836-4
Thomson, R., Hodgman, T. C., Yang, Z. R., & Doyle, A. K. (2003). Characterizing proteolytic
cleavage site activity using bio-basis function neural networks. Bioinformatics, 19 (14),
– 1747. DOI: 10.1093/bioinformatics/btg237
William, T., Jelip, J., Menon, J., Anderios, F., Mohammad, R., Mohammad, T. A. A., ... Barber, B. E.
(2014). Changing epidemiology of malaria in Sabah, Malaysia: increasing incidence of
Plasmodium knowlesi. Malaria Journal, 13 (1), 390. DOI: https://doi.org/10.1186/1475-
-13-390
Yoon, H. R., Kang, C. B., Chia, J., Tang, K., & Yoon, H. S. (2006). Expression, purification, and
molecular characterization of Plasmodium falciparum FK506-binding protein 35
(PfFKBP35). Protein Expression and Purification, 53 (1), 179 – 185. DOI: https://doi.
org/10.1016/j.pep.2006.12.019
Downloads
Published
How to Cite
Issue
Section
Total Views: 374
| 
Total Downloads: 327
