Butane monooxygenase of ‘Pseudomonas butanovora’: purification and biochemical characterization of a terminal-alkane hydroxylating diiron monooxygenase

Abstract

Butane monooxygenase (sBMO) has been purified to homogeneity from the Gram-negative b-proteobacterium 'Pseudomonas butanovora' and confirmed to be a three-component diiron monooxygenase system. The reconstituted enzyme complex oxidized C 3 -C 6 linear and branched aliphatic alkanes, which are growth substrates for 'P. butanovora'. The sBMO complex was composed of an iron-containing hydroxylase (BMOH), a flavo-iron sulfur-containing NADH-oxidoreductase (BMOR) and a small regulatory component protein (BMOB). The physical characteristics of sBMO were remarkably similar to the sMMO family of soluble multicomponent diiron monooxgenases. However, the catalytic properties of sBMO were quantitatively different in regard to inactivation in the presence of substrate and product distribution. BMOH was capable of ethene oxidation when supplied with H 2 O 2 and ethene (known as the peroxide shunt), but this activity was at least three orders of magnitude less than that observed for the hydroxylase of sMMO of Methylosinus trichosporium OB3b. BMOH and BMOR were efficient in the oxidation of ethene in the absence of BMOB with regard to rate of reaction and product yield. Regiospecificity of sBMO was strongly biased towards primary hydroxylation, with ¢80 % of the hydroxylations occurring at the terminal carbon atom.

Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

“…The alternative iron source yielded BMOH that exhibited much tighter elution profiles off the Sephacryl S-300 HR gel-filtration column due to significantly reduced self-aggregation, as determined by dynamic light scattering. Additionally, the iron content of BMOH, as measured by the ferrozine spectrophotometric assay (Percival, 1991), contained 2.1-2.4 irons per active site rather than the 1.4-1.8 reported previously (Dubbels et al, 2007). Typical preparations of the enzyme complex resulted in activities ranging from 400 to 700 nmol min 21 mg 21 .…”

“…Wild-type (WT) T. butanivorans was grown on butane as described previously (Dubbels et al, 2007) with the exception that 100 mM Fe 3+ -EDTA was used as an iron source instead of 500 mM FeSO 4 . 7H 2 O. Mutant strain G113N, which has residue 113 of the a-subunit of BMOH altered from Gly to Asn (Halsey et al, 2006), was grown identically to WT except that 50 mM MnCl 2 and 10 mM Na 2 CO 3 were added to the medium to facilitate the downstream metabolism of secondary alcohols.…”

“…7H 2 O. Mutant strain G113N, which has residue 113 of the a-subunit of BMOH altered from Gly to Asn (Halsey et al, 2006), was grown identically to WT except that 50 mM MnCl 2 and 10 mM Na 2 CO 3 were added to the medium to facilitate the downstream metabolism of secondary alcohols. WT BMOH and BMOR components of sBMO were purified as previously described (Dubbels et al, 2007). The G113N BMOH was purified identically to the WT hydroxylase.…”

“…Additionally, sequence alignment and structural modelling show that all residues directly lining the active site cavity of BMOH are the same as those in the methane monooxygenase hydroxylase (MMOH). Despite the similarities, several biochemical observations indicate that sBMO represents a class distinct from the sMMO family: (i) methanol accumulation ceases during methane oxidation once~20-50 mM has been reached (Halsey et al, 2006), whereas sMMO can continue to accumulate methanol; (ii) sBMO is predominantly a terminal hydroxylator of intermediate-chain-length alkanes (Dubbels et al, 2007), whereas sMMO forms secondary alcohols (Froland et al, 1992); (iii) product regioselectivity is minimally altered by the presence of BMOB (Dubbels et al, 2007), unlike sMMO (Froland et al, 1992); (iv) catalase is required to maintain hydroxylase activity during steady-state turnover (Dubbels et al, 2007) but not to maintain sMMO activity; and (v) the use of peroxide in the 'peroxide shunt' mechanism of substrate oxidation is three orders of magnitude less efficient in sBMO than in sMMO (Dubbels et al, 2007).…”

“…sBMO is a three-component complex that was recently purified and found to have a similar architecture to that of sMMO: (i) a 250 kDa hydroxylase component (butane monooxygenase hydroxylase; BMOH) with (abc) 2 subunit composition that, by analogy with sMMO, contains the diiron active site in the a-subunit; (ii) a 40 kDa flavo-ironsulfur-containing reductase (butane monooxygenase reductase; BMOR) that shuttles electrons from NADH to the active site of BMOH; and (iii) a 15 kDa regulatory protein (butane monooxygenase regulatory component; BMOB) that enhances the overall activity of the complex (Dubbels et al, 2007). Sequence analysis shows BMOH to be most closely related to sMMO (65, 42 and 38 % amino acid identity in the a-, b-and c-subunits of BMOH, respectively) (Sluis et al, 2002).…”