PARENT SESSION
Posters P5C Biosynthesis and assembly: Pigments. Abstracts (643-659)

The pufQ gene regulates activity increases in two enzymes of protoporphyrin synthesis in Rhodobacter capsulatus. Himani Utkhede^*,1, William Richards¹, ¹ Department of Molecular Biology and Biochemistry, Burnaby, BC, Canada

ABSTRACT- We have studied the effect of the presence or absence of the pufQ gene on the activity of two enzymes of protoporphyrin IX synthesis during the transition of Rhodobacter capsulatus from aerobic to semiaerobic growth conditions when increased bacteriochlorophyll synthesis is being induced. When the pufQ gene is disrupted or missing, the activity of coproporphyrinogen III (Coprogen) oxidase (encoded by hemZ) is not increased normally during such transitions. A normal increase cannot be rescued by restoration of the pufQ gene in trans. A previously uncharacterized gene (orf227) has been implicated as encoding an aerobic repressor of hemZ. When an intact pufQ gene is present in its normal chromosomal location, it can relieve the repression of hemZ under semiaerobic conditions, perhaps by down-regulating the expression of orf227. When the pufQ gene is disrupted or missing, the activity of porphobilinogen (PBG) synthase (encoded by hemB) is also not increased normally during transfer from aerobic to semiaerobic growth conditions. However, unlike Coprogen oxidase, a normal increase in activity can be substantially rescued by restoration of the pufQ gene in trans. Also, Orf227 has not been implicated as an aerobic repressor of hemB. The pufQ gene product may, therefore, have a direct effect on the activation of PBG synthase by some other mechanism. A kinetic analysis has confirmed that PBG synthase follows classic Michaelis-Menten kinetics with ALA exhibiting a Michaelis constant of 2.66 mM. Two downstream biosynthetic intermediates, uroporphyrinogen III and coproporphyrinogen III, were found to be potent mixed-type inhibitors of PBG synthase. Concentrations of 0.03 mM of either inhibitor reduced the apparent maximum velocity of PBG synthase by 78% and 69%, respectively. Hence, down-regulation of Coprogen oxidase activity might result in a negative-feedback inhibition of PBG synthase by a subsequent increase in the concentration of coproporphyrinogen III and/or uroporphyrinogen III.

KEY WORDS: biosynthesis, bacteriochlorophyll, regulation, pufQ