A recombinant gamma-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis (rPhGshA II) was produced and characterised. This enzyme catalyses the first step of glutathione biosynthesis by forming gamma-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction. The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of the biosynthesis, was already characterised. rPhGshA II is a monomer of 58 kDa and its activity was characterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was active even at cold temperatures in a moderately alkaline buffer containing a high concentration of Mg++; 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate of rPhGshA II at 15 C was higher than that reported for rPhGshB, thus suggesting that formation of gamma-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. haloplanktis. rPhGshA II had different affinities for its substrates, as evaluated on the basis of the KM values for ATP (0.093 mM), glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of rPhGshA II, probably through the binding to an enzyme pocket different from the active site. Also the oxidised form of glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared to rPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on glutathione biosynthesis in the first cold-adapted source; however, another possible redundant gamma-glutamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of this cellular thiol in P. haloplanktis.

The cold-adapted g-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis

MASULLO, Mariorosario;
2014

Abstract

A recombinant gamma-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis (rPhGshA II) was produced and characterised. This enzyme catalyses the first step of glutathione biosynthesis by forming gamma-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction. The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of the biosynthesis, was already characterised. rPhGshA II is a monomer of 58 kDa and its activity was characterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was active even at cold temperatures in a moderately alkaline buffer containing a high concentration of Mg++; 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate of rPhGshA II at 15 C was higher than that reported for rPhGshB, thus suggesting that formation of gamma-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. haloplanktis. rPhGshA II had different affinities for its substrates, as evaluated on the basis of the KM values for ATP (0.093 mM), glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of rPhGshA II, probably through the binding to an enzyme pocket different from the active site. Also the oxidised form of glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared to rPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on glutathione biosynthesis in the first cold-adapted source; however, another possible redundant gamma-glutamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of this cellular thiol in P. haloplanktis.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11367/29494
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