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

Abstract

NADPH oxidases (NOXs) are membrane enzymes whose sole function is the generation of reactive oxygen species. Humans have seven NOX isoenzymes that feature distinct functions in immune response and cell signaling but share the same catalytic core comprising a FAD-binding dehydrogenase domain and a heme-binding transmembrane domain. We previously described a mutation that stabilizes the dehydrogenase domain of a prokaryotic homolog of human NOX5. The thermostable mutant exhibited a large 19 C increase in the apparent melting temperature (app T m) and a much tighter binding of the FAD cofactor, which allowed the crystallization and structure determination of the domain holo-form. Here, we analyze the transferability of this mutation onto prokaryotic and eukaryotic full-length NOX enzymes. We found that the mutation exerts a significative stabilizing effect on the full-length NOX5 from both Cylindrospermum stagnale (app T m increase of 8 C) and Homo sapiens (app DT m of 2 C). Enhanced thermal stability resulted in more homogeneous preparations of the bacterial NOX5 with less aggregation problems. Moreover, we also found that the mutation increases the overall expression of recombinant human NOX4 and NOX5 in mammalian cells. Such a 2-5-fold increase is mainly due to the lowered cell toxicity, which leads to higher biomasses. Because of the high sequence identity of the catalytic core within this family of enzymes, this strategy can be a general tool to boost the production of all NOXs.

EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

“…Strep tagged-EROS was co-transfected in HEK293-F cells with GFP-gp91 phox and p22 phox . Then, 48 hr post-transfection cells were harvested and cell membranes were prepared as described in Ceccon et al, 2017 . Membranes were solubilised with 1% LMNG in 50 mM HEPES pH 7.5, 100 mM NaCl, 20% (v/v) glycerol, and passed through Streptactin-resin.…”

EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling

“…Strep tagged-EROS was co-transfected in HEK293-F cells with GFP-gp91 phox and p22 phox . 48h post transfection cells were harvested and cell membranes were prepared as described in (Ceccon et al, 2017). Membranes were solubilised with 1% LMNG in 50mM Hepes pH 7.5, 100mM NaCl, 20% (v/v) glycerol, and passed through Streptactin-resin.…”

NADPH Oxidases (NOX): An Overview from Discovery, Molecular Mechanisms to Physiology and Pathology

“…Study of the interaction between oxygen and the external heme suggests that the reduction of O 2 occurs via an outer sphere mechanism [97,105,106] in which oxygen is not directly physically coordinated with the ferric ion of the distal heme but receives the electron by indirect saltatory transfer from the heme periphery. This model is consistent with the insensitivity of the oxidase to classical inhibitors of respiration such as carbon monoxide, azide or cyanide and confirms the absence of a free position on the heme iron.…”