MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases

Abstract

The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins copurify with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.

Figure 1

Purified proteins and assessment of β-Lys activation using ATP/PPi exchange assays. (A) 15% SDS-PAGE/Coomassie blue staining of 2 μg H6-CmnO (lane 1), 3 μg H6-CmnO co-produced with CmnN (lane 2), 2 μg CmnN (lane 3), molecular mass markers (7.1, 20.6, 28.9, 34.8, 49.1, 80.0, 124.0, 209.0 kDa; lane 4), 2 μg H6-VioO (lane 5), 3 μg H6-VioO co-produced with VioN (lane 6), 2 μg CmnN (lane 7). CmnN and VioN in lanes 2 and 6, respectively, were confirmed by mass spectrometry. (B) Comparison of β-Lys activation by 100 nM H6-CmnO alone or with 1.6 μM CmnN and 100 nM H6-VioO alone or with 1.6 μM VioN. Assays were run for 15 min (H6-CmnO-containing) or 1 hr (H6-VioO-containing) with 1 mM β-Lys.

Figure 2

Analysis of ENT production in vivo and Purified EntF. (A) Growth curve comparing wild-type MG1655 to MG1655ΔybdZ with an empty vector (pBAD33) or vector expressing ybdZ (pBAD33+ybdZ). (B) Analysis of purified EntF-H6 overproduced in the presence or absence of YbdZ. 1 μg EntF (lane 1), 1 μg EntF+YbdZ (lane 2), 17 μg EntF (lane 3), 17 μg EntF+YbdZ (lane 4). 10-20% acrylamide gradient gel (left), 15% acrylamide gel (right).