NADPH:protochlorophyllide oxidoreductase from Synechocystis: Overexpression, purification and preliminary characterisation

Abstract

The reduction of the C17-C18 double bond of protochlorophyllide to form chlorophyllide is a key regulatory reaction in the chlorophyll biosynthetic pathway. The reaction is catalysed by the light-dependent enzyme NADPH:protochlorophyllide oxidoreductase (POR). The enzyme is one of only two enzymes in nature known to require light for catalysis, the other being DNA photolyase. POR from the cyanobacterium Synechocystis has been overproduced in Escherichia coli with a hexa-histidine tag at the N-terminus using the pET9-His expression vector. The enzyme was produced in large quantities as a soluble protein that could be purified by a single-step purification procedure on a Ni2+-Sepharose affinity column. The purified enzyme is catalytically active and has facilitated a preliminary characterisation of its kinetic and substrate binding properties.