Biochemical Characterization of Molybdenum Cofactor-free Nitrate Reductase from Neurospora crassa

Abstract

Background: Eukaryotic nitrate reductase maturation is poorly understood. Results: Binding of molybdenum cofactor to apo-nitrate reductase is independent from other prosthetic groups. Conclusion: Active site formation of eukaryotic nitrate reductase is an autonomous process intrinsically tied to nitrate reductase dimerization. Significance: The understanding of molybdenum cofactor-dependent enzyme maturation is of significance as molybdenum enzymes are involved in essential cellular processes.

Enzymatic characterization of recombinant nitrate reductase expressed and purified from Neurospora crassa

“…(Honda and Selker, 2009) by replacing the multiple cloning site and the epitope tag (FLAG-tag) encoding sequences with the Twin-Strep Ò -tag encoding sequence (Schmidt and Skerra, 2007) and a multiple cloning site that has readily been introduced by us into a set of bacterial Twin-Strep-tag Ò expression vectors (Ringel et al, 2013), for details please see Fig. 1C.…”

“…All proteins except for the undiluted negative control protein anti-pentaHIS IgG (Qiagen #35370) were previously transferred to the same buffer and the concentration was determined using a NanoDrop Spectrophotometer at 280 nm and 650 nm. 25 nM of the labeled antibodies were titrated with 16 step-increasing concentrations of Cnx1E Strep-tag Ò II (antigen) generated from 2:1 serial dilutions starting with initial concentrations of 800 nM for 7G8 and 1600 nM for 3E11 or 320 nM and 80 nM for the CcR (cytochrome c reducing fragment) Twin-Strep-tag Ò fusion protein (Ringel et al, 2013) antigen, respectively. After 50 min incubation time the serial dilution was filled into standard capillaries.…”

“…For a typical IP 15 lg of purified monoclonal Strep-tag Ò antibody 7G8 was incubated with 5 -50 lg recombinant Twin-Strep-tag Ò -tagged N. crassa NR purified from E. coli (expression and purification were performed according to (Ringel et al, 2013) in 500 lL Protein A/G IgG binding buffer for 2 h at room temperature. Routinely, 25-50 lL of Pierce Protein A/G magnetic beads (Thermo Scientific) were prepared for use according to manufacturer's instructions and used for IP as follows.…”

“…Routinely, measurements of NR enzymatic activity have been carried out as described earlier (Ringel et al, 2013). For comparison of nitrate reducing activity Mo content from E. coli and N. crassa NR preparations was analyzed by inductively coupled plasma mass spectrometry as previously described (Ringel et al, 2013) and NR activity was correlated to NR Mo centers.…”

“…Epitope tagged proteins are routinely isolated by using tag-specific antibodies, rendering the large scale protein preparation difficult. In previous work, we biochemically characterized the N. crassa nitrate reductase (NR) 1 recombinantly produced in E. coli (Ringel et al, 2013). N. crassa NR is a complex metalloenzyme with an apparent molecular weight of ca.…”