IN-VITRO RECONSTITUTION OF SULFITE REDUCTASE FROM PSEUDOMONAS AERUGINOSA

Abstract

Recent work has established a link between a ferredoxin:NAD(P)H oxidoreductase (FprA) and sulfite assimilation in members of the genus Pseudomonas.  This suggested that FprA is a component of a novel sulfite reductase enzyme.  That hypothesis is consistent with the fact that only one component of the well-characterized E. coli a8?4 sulfite reductase has been identified in Pseudomonas genomes; i.e the ? siroheme subunit CysI is present but not the a flavoprotein subunit CysJ.  This led to the hypothesis that FprA is a component of a novel sulfite reductase enzyme.  Our aim is to test that hypothesis by in-vitro reconstitution using the purified proteins CysI and FprA.  We have successfully overexpressed and purified FprA from Pseudomonas aeruginosa.  The strategy for production of purified CysI has been complicated by the requirement for concomitant expression of CysG (siroheme synthase).  We are also investigating the possibility that a downstream, overlapping reading frame (PA1837) may also be necessary for functional CysI production.