Succinate as anin vitro electron donor for the particulate methane mono-oxygenase ofMethylosinus trichosporium OB3b

Cornish, Alex; MacDonald, James M.; Burrows, K. J.; King, T.S.; Scott, D. H.; Higgins, I. J.

doi:10.1007/bf01030278

Cited by 25 publications

(14 citation statements)

References 6 publications

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“…The 45-kDa subunit is one of the polypeptides observed previously in the electrophoretic profile of the membranes when the organism switches from sMMO to pMMO expression (35)(36)(37)(38)(39). In these experiments, when the organism switches from sMMO to pMMO expression, increasingly higher levels of the 45-and 26-kDa polypeptides were observed concomitantly with decreasing amounts of sMMO hydroxylase-associated polypeptides (35)(36)(37)(38)(39).…”

Section: Characterization Of the Purified Pmmo Hydroxylase Activitymentioning

confidence: 77%

“…In these experiments, when the organism switches from sMMO to pMMO expression, increasingly higher levels of the 45-and 26-kDa polypeptides were observed concomitantly with decreasing amounts of sMMO hydroxylase-associated polypeptides (35)(36)(37)(38)(39). We surmise that the E-clusters reside in the exposed domains of this subunit on the cytoplasmic side.…”

Section: Characterization Of the Purified Pmmo Hydroxylase Activitymentioning

confidence: 98%

“…Copper ions not only regulate the expression of the pMMO but have been found to be crucial for pMMO activity. The expression of the pMMO is accompanied by the formation of an extensive network of intracytoplasmic membranes, where the membrane-bound pMMO resides (35)(36)(37)(38)(39). An increase in carbon to biomass conversion efficiency is also observed.…”

mentioning

confidence: 99%

“…M and Methylomonas methanica 68 -1 (6, 30 -34). In strains capable of expressing either the sMMO or pMMO, the sMMO is expressed under copper stress only (low copper/biomass ratio) (35)(36)(37)(38)(39). Otherwise, the pMMO is expressed.…”

mentioning

confidence: 99%

“…An increase in carbon to biomass conversion efficiency is also observed. Three new polypeptides with apparent molecular masses of 45,35, and 26 kDa were observed in the membrane fractions when M. capsulatus (Bath) switched from expressing the sMMO to the pMMO (35)(36)(37)(38)(39).…”

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confidence: 99%

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The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme

Nguyen

Elliott

Yip

et al. 1998

Journal of Biological Chemistry

220

240

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The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivating Methylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl ␤-D-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60 -80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, ␣, ␤, and ␥, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, ␣, ␤, and ␥, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The ␣ and ␣ subunits from these two forms of the pMMO contain identical N-terminal sequences. The ␥ subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12-15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (ϳ10 H ؉ /94-kDa monomeric unit) and eventually become completely inactive.The enzyme methane monooxygenase, found in methanotrophic bacteria, catalyzes the conversion of methane to methanol using dioxygen as a co-substrate at ambient temperatures and pressures (1, 2). This system has attracted considerable attention, since it provides an ideal natural model to study methane activation and functionalization, a subject of significant current interest (3). Two distinct species of methane monooxygenase (MMO) 1 are known to exist at different cellular locations, a cytoplasmic (soluble) MMO and a membrane-bound (particulate) MMO (4). The soluble MMO (sMMO) is a complex threecomponent system consisting of a hydroxylase, a reductase, and a small regulatory protein (4). The sMMO has been investigated extensively by several research groups (5-21). The xray crystal structure of the sMMO hydroxylase isolated from Methylococcus capsulatus (Bath) has been solved (22, 23). The hydroxylase active site contains a non-heme binuclear iron cluster. In contrast, the particulat...

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Section: Characterization Of the Purified Pmmo Hydroxylase Activitymentioning

confidence: 77%

Section: Characterization Of the Purified Pmmo Hydroxylase Activitymentioning

confidence: 98%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme

Nguyen

Elliott

Yip

et al. 1998

Journal of Biological Chemistry

220

240

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Batch cultivation of Methylosinus trichosporium OB3b. I: Production of soluble methane monooxygenase

Park

Hanna

Taylor

et al. 1991

Biotech & Bioengineering

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Methanotrophs have promising applications in bioremediation and in the production of fuel-related chemicals due to their nonspecific enzyme, methane monooxygenase (MMO). The optimal conditions for cell growth and production of the soluble from of MMO (sMMO) were determined from batch cultivations of an obligatory methanotrophs, Methylosinus trichosporium OB3b, in shake flasks and a 5-L bioreactor. It was confirmed that a copper deficiency is essential for the formation of the cytoplasmic sMNO. Optimum cell growth without added copper was observed at pH 6.0-7.0, temperature of 30-34 degrees C, and phosphate concentration of 10-40 mM. In the bioreactor experiments, external CO(2) addition eliminated the long lag period observed in the absence of added CuSO(4), i.e., prior to the exponential cell growth phase. When methane was continuously supplied, the profile of the cell growth showed two different phases depending on the availability of nitrate, an initial fast exponential growth phase (specific growth rate, micro = 0.08 h(-1)) and a later slow growth phase (micro = 0.008 h(-1)). The cell density at the transition from a fast to a slow growth rate was proportional to the initial medium nitrate concentration in the range 5-20 mM and cell yield was estimated to be 7.14 g dry cell wt/g N. Whole-cell sMNO activity remained essentially constant regardless of the growth rate unit cell growth stopped. With an initial medium iron concentration below 40 mM, an abrupt decrease in sMNO activity was observed. The lower sMNO activity could be restored by supplying additional iron to the bioreactor culture. Cell yield on iron was estimated to be 1.3 x 10(3) g dry cell wt/g Fe.

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Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase

Park

Shah

Taylor

et al. 1992

Biotech & Bioengineering

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The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 microM and remained constant upon further increases of the copper concentration to 120 microM. The cell growth rate (micro), on the other hand, decreased over the entire range (10-120 microM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 microM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.

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Succinate as anin vitro electron donor for the particulate methane mono-oxygenase ofMethylosinus trichosporium OB3b

Cited by 25 publications

References 6 publications

The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme

The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme

Batch cultivation of Methylosinus trichosporium OB3b. I: Production of soluble methane monooxygenase

Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase

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