title: Characterization of a High Affinity Phytochelatin Synthase from The Cd-Utilizing Marine Diatom Thalassiosira pseudonana.
abstract: Phytochelatin synthase (PC synthase) is the enzyme that catalyzes the production of phytochelatins, peptides of the structure (γ-Glu-Cys)n -Gly, where n = 2-11, from the sulfhydryl-containing tripeptide glutathione, in response to elevated metal exposure. Biochemical utilization of Cd in the marine diatom Thalassiosira weissfloggi, as well as unusually high ratios of PC to Cd in some Thalassiosira species including T. pseudonana Hasle et Heimdal, motivated the characterization of T. pseudonana PC synthase 1 (TpPCS1). This enzyme is the product of one of three genes in the T. pseudonana genome predicted to encode for a PC synthase based on its homology to canonical PC synthases previously examined. TpPCS1 was cloned, expressed in Escherichia coli and purified under both aerobic and anaerobic conditions. TpPCS1 exhibits several characteristics that set it distinctly apart from the well-studied PC synthase, Arabidopsis thaliana PCS1 (AtPCS1). It is extremely sensitive to oxidation, which suppresses activity, and it is readily inhibited by the addition of Cd in the absence of thiolate ligands. TpPCS1 also has significantly greater affinity for one of its key substrates, the bis-glutathionato-Cd complex. TpPCS1 kinetics is best described by a ternary complex model, as opposed to the ping-pong model used to describe AtPCS1 kinetics. The findings indicate that although the function of TpPCS1 is synonymous to that of AtPCS1, its divergent biochemistry suggests adaptation of this enzyme to the distinct trace metal chemistry of the marine environment and the unique physiological needs of T. pseudonana.
authors.affiliation: Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York, 14853, USA.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Gupton-Campolongo
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, New York, 14853, USA.
authors.forename: Leonardo M
authors.initials: LM
authors.lastname: Damasceno
authors.affiliation: Department of Microbiology, Cornell University, Ithaca, New York, 14853, USA.
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Hay
authors.affiliation: Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York, 14853, USA.
authors.forename: Beth A
authors.initials: BA
authors.lastname: Ahner
keywords: Thalassiosira pseudonana; cadmium; metal detoxification; phytochelatin; phytochelatin synthase