Domain organization of phytochelatin synthase: functional properties of truncated enzyme species identified by limited proteolysis.
Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms. No structural information on this enzyme is as yet available. It is generally believed, however, that the active site region is located in the more conserved N-terminal portion of PCS, whereas various, as yet unidentified (but supposedly less critical) roles have been proposed for the C-terminal region. To gain insight into the structural/functional organization of PCS, we have conducted a limited proteolysis analysis of the enzyme from Arabidopsis (AtPCS1), followed by functional characterization of the resulting polypeptide fragments. Two N-terminal fragments ending at positions 372 (PCS_Nt1) and 283 (PCS_Nt2) were produced sequentially upon V8 protease digestion, without any detectable accumulation of the corresponding C-terminal fragments. As revealed by the results of in vivo and in vitro functional assays, the core PCS_Nt2 fragment is biosynthetically active in the presence of cadmium ions and supports phytochelatin formation at a rate that is only approximately 5-fold lower than that of full-length AtPCS1. The loss of the C-terminal region, however, substantially decreases the thermal stability of the enzyme and impairs phytochelatin formation in the presence of certain heavy metals (e.g. mercury and zinc, but not cadmium or copper). The latter phenotype was shared by PCS_Nt2 and by its precursor fragment PCS_Nt1, which, on the other hand, was almost as stable and biosynthetically active (in the presence of cadmium) as the full-length enzyme. AtPCS1 thus appears to be composed of a protease-resistant (and hence presumably highly structured) N-terminal domain, flanked by an intrinsically unstable C-terminal region. The most upstream part of such a region (positions 284-372) is important for enzyme stabilization, whereas its most terminal part (positions 373-485) appears to be required to determine enzyme responsiveness to a broader range of heavy metals.
Ruotolo
Roberta
R
Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, 43100 Parma, Italy.
Peracchi
Alessio
A
Bolchi
Angelo
A
Infusini
Giuseppe
G
Amoresano
Angela
A
Ottonello
Simone
S
D019881:Aminoacyltransferases / Q000737:chemistry*; D017360:Arabidopsis / Q000378:metabolism; D001665:Binding Sites; D001669:Biochemical Phenomena; D001671:Biochemistry; D002104:Cadmium / Q000737:chemistry; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005978:Glutathione; D015151:Immunoblotting; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D054811:Phytochelatins; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013997:Time Factors
D018076:DNA, Complementary; D008667:Metalloproteins; D010455:Peptides; D011506:Proteins; D002104:Cadmium; D054811:Phytochelatins; D019881:Aminoacyltransferases; C093784:glutathione gamma-glutamylcysteinyltransferase; D005978:Glutathione; D003545:Cysteine