Article Figures & Data
Figures
- FIG. 1.
Identification of proteins required for cyanide oxygenase activity in P. fluorescens. (A) Resolution of CNO into four active protein fractions (P1 to P4) by gel filtration chromatography. Proteins (1 mg) were loaded onto a Superdex 200 column (30 cm by 10 mm) and eluted with 20 mM Na2HPO4 buffer (pH 7.0) at 0.5 ml per min. (B) The CNO activity of reconstituted protein components recovered as described for panel A was measured either as the relative amount of radioactive K14CN converted to 14CO2 when supplemented with formate dehydrogenase (+FDH) or the amount of unlabelled CN converted to ammonia (or formate) in its absence (−FDH). In each case, reactions (0.25 ml) were conducted in sealed vials at 30oC and supplied with ∼24 μg protein (equal component proportions), 0.1 mM KCN, 0.2 mM NADH, and 0.5 μM dihydrobiopterin (H2B) in 50 mM Na-K phosphate buffer (pH 7.0). Reactions mixtures supplied with FDH (2 mU) contained 1 μCi K14CN (48 KBq, ∼100 μM) and were incubated for 1 h before 14CO2 was quantified versus those from which FDH was omitted, in which case ammonia (or formate) was determined after 10 min. (C) SDS-PAGE (12%) analysis of CNO components. Lanes: Std., molecular size standards (5 μg); H, crude extract (5 μg); B, source 30Q fraction (5 μg); and P1 to P4 (10 μg each).
- FIG. 2.
Individual activities of P1 to P4 protein components of CNO. Enzymes were assayed as described in Materials and Methods. P3-containing reaction mixtures were supplied 17.5 μg of protein. Data sets are those from at least two independent experiments.
- FIG. 3.
Gel filtration chromatography of P3 (CynDPf11764) at pH 8.0. P3 (20 μg protein) was applied to a Superdex 200 column, and the molecular mass of eluted proteins was determined against calibrated molecular mass standards. Inset, native PAGE analysis of CynDPf11764 (peak at 300 kDa) recovered from gel filtration and molecular mass standards (Std).
Tables
- TABLE 1.
Activity-based purification of cyanide oxygenase from P. fluorescens NCIMB 11764
Purifi- cation stepa Total protein (mg) Volume (ml) Total activityb (mU) Sp act (mU mg−1) Purifi- cation (fold) Recovery (%) Fraction H 330 8 2,640 8 1 100 Source 30Q 1 1 500 500 71 18 Superdex 200 0.98 0.5 450 900 112 17 ↵ a Fraction H, high-molecular weight retentate recovered after passing 150,000 × g high-speed supernatant through a 10-kDa mwco ultraconcentrator; Source 30Q, fraction recovered after anion-exchange chromatography; Superdex 200, combined P1 to P4 fractions recovered after gel filtration chromatography.
↵ b CNO activity was determined by measuring O2 or NADH consumption at 30°C in reaction mixtures (0.5 ml) supplied with 0.1 mM KCN, 0.2 mM NADH, and 0.5 μM dihydrobiopterin in 50 mM Na-K phosphate buffer (pH 7.0). The amounts of protein were as follows: 0.5 to 1 mg ml−1 for Fraction H and Source 30Q and 50 μg ml−1 and for Superdex 200 fractions equally proportioned between P1, P2, P3, and P4.
- TABLE 2.
Components of cyanide oxygenase from P. fluorescens NCIMB 11764
Analysis P1 P2 P3 P4 Enzyme NADH oxidase (Nox) NADH peroxidase (Npx) Cyanide dehydratase (CynD) Carbonic anhydrase (CA) E.C. no. 1.6.99.3 1.11.1 3.5.5.- 4.2.1.1 Sp act (U/mg)a 1 1 100 2000 Molecular mass (kDa) SDS-PAGE 76b (46, 31) 47 38 (43c) 29 Mass spectrometry NDe 49.7 ND 28.9 Gel filtrationd 230 100 ≥300 58 Suggested composition α3β3 α2 Unknown α2 N-terminal sequence ND MKVIVLGSSHGGYEAVEELLNLHPD ND N-blocked Tryptic peptide sequence ND ND ND LVQFHFHWGS YAAELHL ↵ a Defined as 1 μmol of substrate converted per min.
↵ b Possible dimer of 46- and 31-kDa species.
↵ c Internal peptide from trypic digest shows 100% amino acid sequence homology (VQDPLEIVGLR) to elongation factor Tu from P. putida.
↵ d Determined at pH 7.0, except for CynD, which was performed at pH 8.0.
↵ e ND, not determined.
