Iron, an essential element for biosynthesis of aromatic compounds

Area of Science:

• Biochemistry
• Enzymology
• Metalloprotein characterization

Background:

• 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHP synthase) is a key enzyme in the shikimate pathway.
• Previous studies suggested the presence of cobalt in DAHP synthase, but this has not been definitively confirmed.
• The enzyme-substrate complex with phosphoenolpyruvate (PEP) is crucial for enzyme activity.

Purpose of the Study:

• To determine the metal cofactor composition of homogeneous DAHP synthase from Escherichia coli.
• To investigate the role of the metal cofactor and substrates in the enzyme's spectral properties and activity.
• To clarify the discrepancy regarding cobalt presence in previous research.

Main Methods:

• Atomic absorption analysis to quantify metal content.
• Spectrophotometry to determine absorption maxima and extinction coefficients.
• Enzyme assays and denaturation studies using guanidine-hydrochloride and substrate manipulation (erythrose 4-phosphate).

Main Results:

• Homogeneous DAHP synthase preparations contain approximately one mole of iron per mole of native enzyme.
• No cobalt was detected, contradicting earlier suggestions.
• A unique absorption maximum at 350 nm (ε ≈ 3500 M⁻¹cm⁻¹) was observed for the enzyme-phosphoenolpyruvate complex.
• The 350 nm band and enzyme activity were lost upon denaturation or removal of PEP, but iron remained bound.
• Iron remained bound to the enzyme even after removal of PEP by erythrose 4-phosphate.

Conclusions:

• Iron is the essential metal cofactor in Escherichia coli DAHP synthase.
• The enzyme-phosphoenolpyruvate complex exhibits a distinct spectral signature at 350 nm, linked to enzyme activity.
• The iron cofactor is stably associated with the enzyme, even when the substrate PEP is removed.