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4 over 25 min, 10 ml/min, monitor at 280 nm), and desalted. After removal of methanol on a rotary evaporator, the sample was lyophilized to give the aa-S-CoA (50 to 80% yield) as a white solid.
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17
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0345350365
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note
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PheATE, PheAT, PheTE, and PheT are deletion mutants or derivatives of the phenylalanine (Phe)-activating enzyme GrsA, containing combinations of the A domain, the T domain, and the epimerization (E) domain. ProCAT is the recombinant first module of TycB, containing a proline (Pro)-activating A domain, a T domain, and the upstream C domain.
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0344919134
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3H]Phe-transfer assay. His-tagged and untagged PheATE are enzymatically identical.
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19
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0345350364
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-1). Nvoc-D-Ala-S-CoA (P, 20:27:0.7), Nvoc-L-Ala-S-CoA (P, 18:20:0.9), Nvoc-D-Phe-S-CoA (P, 18:13:1.4) and (A, 16:9:1.8). D-Phe-S-CoA (P, 15:8:1.9), Nvoc-L-Phe-S-CoA (P, 17:17:1), L-Phe-S-CoA (P, 22:19:1.2). For PheAT acceptor: Nvoc-D-Phe-S-CoA (A, 17:16:1.1). For PheTE acceptor: Nvoc-D-Phe-S-CoA (A, 15:16:0.9).
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0032575648
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0344056794
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note
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3H]Pro (24.7 Ci/mmol). Simultaneously, a 1 μM solution of the other aminoacylated, module in MES buffer, pH 8.0, was also incubated at 37°C After 3 min, the condensation reaction was initiated by combining equal volumes of both solutions. At various time points, 100-μl samples were taken and immediately quenched by the addition of 0.8 ml of 10% trichloroacetic acid (TCA) (w/v) and 20 μl of bovine serum albumin (BSA) solution [2% (w/v)]. The TCA precipitate was washed once with 0.5 ml of 10% TCA (w/v), and the acid-stable label was quantified by LSC. The supernatants were extracted with 0.5 ml of butanol/chloroform [4:1; (v/v)], the organic layers were washed once with 0.5 ml of 0.1 M NaCl, and the amount of extractable label (DKP) was quantified by LSC (15).
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0344056795
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note
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-1.
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0345350361
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Supported in part by NIH grant GM20011, an European Molecular Biology Organization fellowship (T.S.) and a fellowship from the Jane Coffin Childs Memorial Fund for Medical Research (P.J.B.).
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