chlD gene function in molybdate activation of nitrate reductase.

Area of Science:

• Microbiology
• Molecular Biology
• Enzyme Kinetics

Background:

• Escherichia coli nitrate reductase is essential for anaerobic respiration.
• The chlD gene's role in nitrate reductase biosynthesis is not fully understood.

Purpose of the Study:

• To elucidate the specific functions of the chlD gene in nitrate reductase formation in E. coli.
• To investigate the mechanism of molybdenum insertion into nitrate reductase.

Main Methods:

• Analysis of chlD mutants under varying molybdate and tungstate conditions.
• Electrophoretic and radioisotope labeling (99-Mo, 185-W) to study enzyme protein and metal incorporation.
• Enzyme activity assays to quantify nitrate reductase levels.

Main Results:

• chlD mutants form inactive nitrate reductase protein but require molybdate for activation.
• Tungstate supplementation leads to accumulation of inactive enzyme, which can be activated by molybdate.
• The chlD gene product facilitates molybdate insertion at physiological concentrations, with high molybdate bypassing this function.

Conclusions:

• The chlD gene has two roles: promoting enzyme protein accumulation and facilitating molybdenum insertion for active nitrate reductase.
• Molybdenum incorporation is a critical step regulated by the chlD gene product.
• High molybdate concentrations can overcome the chlD requirement through a mass action effect.