TABLE 2.
Product yields from assays of dialyzed cell extracts of m-xylene-grown Azoarcus sp. strain Ta
| Substrate added
|
Product (nmol)b
|
Product labeling | |||||||
|---|---|---|---|---|---|---|---|---|---|
| Aromatic compound | Fumarate | Succinyl-CoA | CoA | ATP | Nitrate | 3-MeBS | E-3-MePI | 3-Methylbenzoate | |
| m-Xylene-d6 (360 nmol, total) | + | − | − | − | + | 200 | NDc | ND | Deuterium labeled |
| m-Xylene-d6 (360 nmol, total) | + | + | − | − | + | 73 | 85 | ND | Deuterium labeled |
| m-Xylene-d6 (360 nmol, total) | + | − | + | + | + | 67 | 49 | ND | Deuterium labeled |
| m-Xylene-d6 (360 nmol, total) | + | − | + | − | + | 130 | ND | ND | Deuterium labeled |
| 3-MeBS (150 nmol, total) | − | − | − | − | + | 110 | ND | ND | |
| 3-MeBS (150 nmol, total) | − | + | − | − | + | 64 | 40 | ND | |
Each anoxic assay mixture (total volume, 1 ml) contained DTT (1 mM) as a reductant and dialyzed cell extracts (ca. 3 mg of protein). Initial concentrations of other substrates added are given as follows: fumarate, 500 μM; succinyl-CoA, 300 μM; CoA, 300 μM; ATP, 2 mM; and nitrate, 2 mM. A total of 360 nmol of m-xylene is equivalent to a liquid concentration of 170 μM. Assay mixtures were incubated for 1 h. Reaction products were extracted, derivatized to methyl esters, and analyzed by GC/MS.
3-MeBS and 3-methylbenzoate were identified by GC/MS by using authentic standards. E-3-MePI was tentatively identified based on mass spectral similarity to an authentic E-PI standard. Assays were conducted in duplicate. Product yields of duplicate assays were typically within 5 to 20% of each other. Product yields shown are averages of the duplicate assays and are representative of other experiments conducted.
ND, not detected. The detection limits of E-3-MePI and 3-methylbenzoate were less than 1 nmol.