Evolutionary and structural analyses of the NADPH oxidase family in eukaryotes reveal an initial calcium dependency

Massari, Marta; Nicoll, Callum R.; Marchese, Sara; Mattevi, A.; Mascotti, María Laura

doi:10.1016/j.redox.2022.102436

Cited by 13 publications

(8 citation statements)

References 69 publications

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“…In strong correlation with the affinity data obtained for the flavins shown in Table 2 , the DH domain mainly interacts with the isoalloxazine ring through polar contacts with the protein backbone and a few residues (P234, F235 and S23) belonging to the conserved FAD-1 binding motif (Massari et al, 2022 ) (see Figure S1). The DH domain shows almost no contact with the ribitol and adenosine parts of the FAD.…”

Section: High Resolution Structure Of the Spnox Dehydrogenase Domainsupporting

confidence: 76%

Section: Production Of Spnox and Spnox Dh Domainmentioning

confidence: 98%

“…SpNOX shares only about 25% identity with the human NOX homologs; nonetheless the NOX family members share several well-conserved motifs identified in eukaryotic NOX by Massari et al (Massari et al, 2022 ). Figure S1A shows the logos corresponding to the eukaryotic NOX motifs in (Massari et al, 2022 ), but constructed from our alignment containing about half prokaryotic sequences. Other than the motif identified as NADPH3, for which our joint prokaryotic-eukaryotic NOX alignment lacked consensus, SpNOX (and other prokaryotic NOX) recognizably share all the motifs identified by Massari & al (Massari et al, 2022 ).…”

Section: Production Of Spnox and Spnox Dh Domainmentioning

confidence: 98%

“…Figure S1A shows the logos corresponding to the eukaryotic NOX motifs in (Massari et al, 2022 ), but constructed from our alignment containing about half prokaryotic sequences. Other than the motif identified as NADPH3, for which our joint prokaryotic-eukaryotic NOX alignment lacked consensus, SpNOX (and other prokaryotic NOX) recognizably share all the motifs identified by Massari & al (Massari et al, 2022 ). The C-terminal motif identified by our alignment includes several residues past the C-terminal end of the motif identified from eukaryotes, indicating the high frequency of additional residues in prokaryotic NOX.…”

Section: Production Of Spnox and Spnox Dh Domainmentioning

confidence: 99%

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X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

Petit-Hartlein,

Vermot,

Thepaut

et al. 2024

eLife

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NADPH oxidases (NOX) are transmembrane proteins that professionally produce reactive oxygen species (ROS) and are distributed widely in both eukaryotes and prokaryotes. Eukaryotes use the ROS products for innate immune defense and signaling; the seven human isoforms of NOX participate in critical physiological and pathophysiological processes. Recently solved structures of two human NOX isoforms provide much new information, but do not fully elucidate controls on the electron transfer pathway from NAD(P)H substrate through FAD and heme cofactors to the final ROS product. SpNOX, a bacterial NOX homolog from Streptococcus pneumoniae , shows robust constitutive activity in detergent solution, making it a good prototype for exploring electron transfer in the NOX family. Here we present crystal structures of wildtype and mutant full-length and dehydrogenase (DH) domain-only constructs of SpNOX. The isolated DH domain acts as a flavin reductase, and both DH and full-length constructs use either NADPH or NADH as substrate. Our data supports hydride transfer from NAD(P)H to FAD as the rate limiting step in electron transfer. Using the DH domain we demonstrate the role of F397 in allowing access of nicotinamide to the flavin isoalloxazine, while in the full length construct we used mutants and flavin analogs to confirm the contribution of both domains to flavin binding observed in the structure. Comparison with homologous enzymes suggests distal heme access may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity of a given NOX family member. Based on this comparison, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.

show abstract

Section: High Resolution Structure Of the Spnox Dehydrogenase Domainsupporting

confidence: 76%

Section: Production Of Spnox and Spnox Dh Domainmentioning

confidence: 98%

Section: Production Of Spnox and Spnox Dh Domainmentioning

confidence: 98%

Section: Production Of Spnox and Spnox Dh Domainmentioning

confidence: 99%

See 2 more Smart Citations

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

Petit-Hartlein,

Vermot,

Thepaut

et al. 2024

eLife

View full text Add to dashboard Cite

show abstract

“…SpNOX shares only about 25% identity with the human NOX homologs; nonetheless the NOX family members share several well-conserved motifs identified in eukaryotic NOX by Massari et al (Massari et al, 2022). Figure S1A shows the logos corresponding to the eukaryotic NOX motifs in (Massari et al, 2022), but constructed from our alignment containing about half prokaryotic sequences. Other than the motif identified as NADPH3, for which our joint prokaryotic-eukaryotic NOX alignment lacked consensus, SpNOX (and other prokaryotic NOX) recognizably share all the motifs identified by Massari & al (Massari et al, 2022).…”

Section: Resultsmentioning

confidence: 99%

X-Ray Structure and enzymatic study of a Bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

Petit-Härtlein,

Vermot,

Thépaut

et al. 2023

Preprint

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NADPH oxidases (NOX) are transmembrane proteins that professionally produce reactive oxygen species (ROS) and are distributed widely in both eukaryotes and prokaryotes. Eukaryotes use the ROS products for innate immune defense and signaling; the seven human isoforms of NOX participate in critical physiological and pathophysiological processes. Recently solved structures of two human NOX isoforms provide much new information, but do not fully elucidate controls on the electron transfer pathway from NAD(P)H substrate through FAD and heme cofactors to the final ROS product. SpNOX, a bacterial NOX homolog from Streptococcus pneumoniae, shows robust constitutive activity in detergent solution, making it a good prototype for exploring electron transfer in the NOX family. Here we present crystal structures of wildtype and mutant full-length and dehydrogenase (DH) domain-only constructs of SpNOX. The isolated DH domain acts as a flavin reductase, and both DH and fulllength constructs use either NADPH or NADH as substrate. Our data supports hydride transfer from NAD(P)H to FAD as the rate limiting step in electron transfer. Using the DH domain we demonstrate the role of F397 in allowing access of nicotinamide to the flavin isoalloxazine, while in the full length construct we used mutants and flavin analogs to confirm the contribution of both domains to flavin binding observed in the structure. Comparison with homologous enzymes suggests distal heme access may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity of a given NOX family member. Based on this comparison, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2 requirement for activation.

show abstract

Nox5: Molecular Regulation and Pathophysiology

Camargo

Montezano

Touyz

2023

NADPH Oxidases Revisited: From Function to Structure

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Evolutionary and structural analyses of the NADPH oxidase family in eukaryotes reveal an initial calcium dependency

Cited by 13 publications

References 69 publications

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

X-Ray Structure and enzymatic study of a Bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

Nox5: Molecular Regulation and Pathophysiology

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