Engineering stability in NADPH oxidases: A common strategy for enzyme production

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• NADPH oxidases (NOXs) are crucial membrane enzymes generating reactive oxygen species, involved in immune response and cell signaling.
• Seven human NOX isoenzymes share a conserved catalytic core, presenting a target for biotechnological manipulation.
• Previous work stabilized a prokaryotic NOX dehydrogenase domain, enabling structural studies.

Purpose of the Study:

• To investigate the transferability of a stabilizing mutation to full-length prokaryotic and eukaryotic NOX enzymes.
• To assess the impact of this mutation on the thermal stability, homogeneity, and expression of NOX enzymes.
• To establish a general strategy for enhancing the production of NOX family proteins.

Main Methods:

• Site-directed mutagenesis to introduce a previously identified stabilizing mutation into full-length NOX enzymes.
• Thermal shift assays to determine the apparent melting temperature (app T_m) of wild-type and mutant NOX enzymes.
• Recombinant protein expression in bacterial and mammalian cell systems, followed by analysis of protein homogeneity, aggregation, and expression levels.

Main Results:

• The mutation significantly increased the thermal stability of full-length NOX5 from *Cylindrospermum stagnale* (8°C) and *Homo sapiens* (2°C).
• Enhanced stability led to more homogeneous preparations of bacterial NOX5 with reduced aggregation.
• Recombinant expression of human NOX4 and NOX5 in mammalian cells increased 2-5 fold, attributed to decreased cell toxicity and higher biomass.

Conclusions:

• The stabilizing mutation is transferable to full-length NOX enzymes, improving their biophysical properties.
• This strategy offers a valuable tool for boosting the recombinant production of various NOX family members.
• The findings pave the way for more efficient research and biotechnological applications of NOX enzymes.