Abstract
We investigated the role of the cofactor PLP and its binding domain in stability and subunit assembly of phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica. Presence of cofactor influences the tertiary structure of EhPSAT because of the significant differences in the tryptophan microenvironment and proteolytic pattern of holo- and apo-enzyme. However, the cofactor does not influence the secondary structure of the enzyme. Stability of the protein is significantly affected by the cofactor as holo-enzyme shows higher T m and C m values for thermal and GdnHCl-induced denaturation, respectively, when compared to the apo-enzyme. The cofactor also influences the unfolding pathway of the enzyme. Although urea-dependent unfolding of both holo- and apo-EhPSAT is a three-state process, the intermediates stabilized during unfolding are significantly different. For holo-EhPSAT a dimeric holo-intermediate was stabilized, whereas for apo-EhPSAT, a monomeric intermediate was stabilized. This is the first report on stabilization of a holo-dimeric intermediate for any aminotransferase. The isolated PLP-binding domain is stabilized as a monomer, thus suggesting that either the N-terminal tail or the C-terminal domain of EhPSAT is required for stabilization of dimeric configuration of the wild-type enzyme. To the best of our knowledge, this is a first report investigating the role of PLP and various protein domains in structural and functional organization of a member of subgroup IV of the aminotransferases.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- PSAT:
-
Phosphoserine aminotransferase
- PLP:
-
Pyridoxal-5′-phosphate
- Ni-NTA:
-
Nickel nitrilotriacetic acid
- SEC:
-
Size exclusion chromatography
References
Akhtar MS, Ahmad A, Bhakuni V (2002) Guanidinium chloride- and urea-induced unfolding of the dimeric enzyme glucose oxidase. Biochemistry 41:3819–3827
Ali V, Nozaki T (2006) Biochemical and functional characterization of phosphoserine aminotransferase from Entamoeba histolytica, which possesses both phosphorylated and non-phosphorylated serine metabolic pathways. Mol Biochem Parasitol 145:71–83
Andrade MA, Chacon P, Merelo JJ, Moran F (1993) Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng 6:383–390
Bollen YJ, Nabuurs SM, van Berkel WJ, van Mierlo CP (2005) Last in, first out: the role of cofactor binding in flavodoxin folding. J Biol Chem 280:7836–7844
Cai K, Schirch D, Schirch V (1995) The affinity of pyridoxal 5′-phosphate for folding intermediates of Escherichia coli serine hydroxymethyltransferase. J Biol Chem 270:19294–19299
Choi YS, Han SK, Kim J, Yang JS, Jeon J, Ryu SH, Kim S (2010) ConPlex: a server for the evolutionary conservation analysis of protein complex structures. Nucleic Acids Res 38:W450–W456
Deu E, Kirsch JF (2007a) Cofactor-directed reversible denaturation pathways: the cofactor-stabilized Escherichia coli aspartate aminotransferase homodimer unfolds through a pathway that differs from that of the apoenzyme. Biochemistry 46:5819–5829
Deu E, Kirsch JF (2007b) The unfolding pathway for Apo Escherichia coli aspartate aminotransferase is dependent on the choice of denaturant. Biochemistry 46:5810–5818
Deu E, Dhoot J, Kirsch JF (2009) The partially folded homodimeric intermediate of Escherichia coli aspartate aminotransferase contains a “molten interface” structure. Biochemistry 48:433–441
Greene RF Jr, Pace CN (1974) Urea and guanidine hydrochloride denaturation of ribonuclease, lysozyme, alpha-chymotrypsin, and beta-lactoglobulin. J Biol Chem 249:5388–5393
Herold M, Leistler B, Hage A, Luger K, Kirschner K (1991) Autonomous folding and coenzyme binding of the excised pyridoxal 5′-phosphate binding domain of aspartate aminotransferase from Escherichia coli. Biochemistry 30:3612–3620
Hester G, Stark W, Moser M, Kallen J, Markovic-Housley Z, Jansonius JN (1999) Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate. J Mol Biol 286:829–850
Jansonius JN (1998) Structure, evolution and action of vitamin B6-dependent enzymes. Curr Opin Struct Biol 8:759–769
Kapetaniou EG, Thanassoulas A, Dubnovitsky AP, Nounesis G, Papageorgiou AC (2006) Effect of pH on the structure and stability of Bacillus circulans ssp. alkalophilus phosphoserine aminotransferase: thermodynamic and crystallographic studies. Proteins 63:742–753
Knapp JA, Pace CN (1974) Guanidine hydrochloride and acid denaturation of horse, cow, and Candida krusei cytochromes c. Biochemistry 13:1289–1294
Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York
Makhatadze GI, Privalov PL (1992) Protein interactions with urea and guanidinium chloride. A calorimetric study. J Mol Biol 226:491–505
Mehta PK, Hale TI, Christen P (1993) Aminotransferases: demonstration of homology and division into evolutionary subgroups. Eur J Biochem 214:549–561
Mishra V, Ali V, Nozaki T, Bhakuni V (2010) Entamoeba histolytica Phosphoserine aminotransferase (EhPSAT): insights into the structure-function relationship. BMC Res Notes 3:52
Muralidhara BK, Wittung-Stafshede P (2005) FMN binding and unfolding of Desulfovibrio desulfuricans flavodoxin: “hidden” intermediates at low denaturant concentrations. Biochim Biophys Acta 1747:239–250
Nandi PK, Robinson DR (1984) Effects of urea and guanidine hydrochloride on peptide and nonpolar groups. Biochemistry 23:6661–6668
Pant K, Crane BR (2005) Structure of a loose dimer: an intermediate in nitric oxide synthase assembly. J Mol Biol 352:932–940
Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612
Satterthwait AC, Jencks WP (1974) The mechanism of the aminolysis of acetate esters. J Am Chem Soc 96:7018–7031
Schellman JA (2002) Fifty years of solvent denaturation. Biophys Chem 96:91–101
Schneider G, Kack H, Lindqvist Y (2000) The manifold of vitamin B6 dependent enzymes. Structure 8:R1–R6
Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31:3381–3385
Singh K, Bhakuni V (2008) Toxoplasma gondii ferredoxin-NADP + reductase: role of ionic interactions in stabilization of native conformation and structural cooperativity. Proteins 71:1879–1888
Wardell SE, Kwok SC, Sherman L, Hodges RS, Edwards DP (2005) Regulation of the amino-terminal transcription activation domain of progesterone receptor by a cofactor-induced protein folding mechanism. Mol Cell Biol 25:8792–8808
West SM, Price NC (1989) The unfolding and refolding of cytoplasmic aspartate aminotransferase from pig heart. Biochem J 261:189–196
Acknowledgments
V.M. is grateful to the Council of Scientific and Industrial Research, Government of India, for the award of a research fellowship. Finally, we are thankful to Dr. Sohail Akhtar, Dr. Prabodh Kapoor and P. Shah for useful suggestions and for generating structural figures. This is communication no. 7996 from CDRI, India.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Mishra, V., Ali, V., Nozaki, T. et al. Biophysical characterization of Entamoeba histolytica phosphoserine aminotransferase (EhPSAT): role of cofactor and domains in stability and subunit assembly. Eur Biophys J 40, 599–610 (2011). https://doi.org/10.1007/s00249-010-0654-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-010-0654-3