Fig. 5

Functional dissection of NosN and NosK. a Extracted ion chromatograms (EICs) of [M-H]⁻ = 188.1 (corresponding to DMIA, 11) for (i) control reaction with the supernatant of boiled NosN, (ii) overnight reaction with 100 μM MIA, 500 μM ATP, 10 μM NosI, 100 μM NosJ and 100 μM NosN that was subsequently treated with 0.2 M NaOH for 30 min, (iii) DMIA synthetic standard and the culture extracts from (iv) S. actuosus wild-type strain, (v) the nosI-knockout mutant and (vi) the nosK-knockout mutant. b A time course analysis of NosK-catalyzed MIA and DMIA release from the NosJ-bound thioesters. The reaction was performed by addition of 10 μM NosK or NosI to the solutions containing 100 μM MIA-NosJ or DMIA-NosJ (which were produced by treating apo-NosJ with Sfp and the corresponding CoA thioesters) and the reaction was quenched by addition of an equal volume of methanol at different time points. MIA and DMIA was quantified by HPLC with UV detection at 300 nm. The reaction was performed in triplicates and the SD are shown by the error bars. It should be noted that NosK also hydrolyzes the CoA-bound MIA and DMIA thioesters with comparative efficiencies with that of the NosJ-bound thioesters