MbtH-Like Proteins as Integral Components of Bacterial Nonribosomal Peptide Synthetases

Abstract

The biosynthesis of many natural products of clinical interest involves large, multi-domain 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 co-purify together with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.Nonribosomal peptide synthetases (NRPSs) are involved in the assembly of natural products of clinical interest such as the antibacterial drugs vancomycin, daptomycin, and capreomycin. A basic understanding how NRPSs catalyze the assembly of such molecules from simple precursors has been established (1). During assembly, each precursor is activated, covalently tethered to the NRPS, and then directionally condensed into the growing molecule by a set of catalytic domains grouped together as modules. Each module is typically composed of an adenylation (A) domain that recognizes and activates each precursor and tethers them to a peptidyl carrier protein (PCP) domain as a thioester. Condensation (C) domains subsequently catalyze directional bond formation between two PCP-linked precursors. Additional domains can add functionality to the precursors or govern its release from the NRPS. The repeating domain/modular structure of NRPSs provides an assembly line-like logic to the biosynthesis of the associated natural products.In bacteria, many gene clusters coding for the NRPS involved in the production of natural products also code for a small (∼70 amino acid) protein containing three conserved tryptophan residues. These proteins have been named the MbtH-like protein superfamily based on their similarity to MbtH from the mycobactin biosynthesis gene cluster (2). The production of some NRPS-dependent natural products requires an MbtH-like protein (3,4), but how these proteins influence production is unknown. Co-production of an MbtH-like protein with an NRPS component enhances protein production levels (5). A direct role in catalysis has been questioned by a report that the enterobactin (ENT) NRPS is functional in vitro in the absence of the associated MbtH-like protein (6). Structural work on the MbtHlike protein from the pyroverdine system (3) and MbtH itself (7) did not reveal any motifs suggestive of a catalytic site, instead, a role in protein-protein interactions.We are investigating the biosynthesis of the antituberculosis drugs capreomycin (CMN) and viomycin (VIO) to better understand NRPS enzymology and develop new derivatives of these drugs using combinatorial biosynthesis. These structurally related non-ribosomal * To whom correspondence should be addressed. Phone: (608) Figure S1, Supporting Information) (8, 9). In addition to the NRPS components, the associated gene clusters code for MbtH-like proteins. This provided us with two related NRPS systems to address questions co...

Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis

“…Felnagle et al (6) have shown that purified YbdZ can activate adenylating enzymes of different NRPSs, albeit with low efficiency. To exclude the possibility that YbdZ would copurify with the expressed adenylating enzymes and interfere with subsequent investigations, we deleted the ybdZ gene from the E. coli expression host utilizing the same Red/ET-mediated recombination strategy as described in a previous study of our group (11).…”

“…Coexpression of CloH and CloY-Felnagle et al (6) reported previously that the adenylating enzymes CmnO and VioO showed moderate activity upon addition of the MbtH-like proteins CmnN and VioN. However, much higher activity could be obtained when CmnO or VioO (as N-terminally His-tagged proteins) was expressed simultaneously with CmnN or VioN (as untagged proteins), followed by copurification of the proteins in the form of the resulting complexes.…”

“…Of the five adenylation domains that activate the different amino acids required for capreomycin biosynthesis, three were dependent on the presence of the MbtH-like protein CmnN, whereas the two others were not. The reason for this difference is unknown (6). The heterologous expression of adenylating enzymes in Escherichia coli was found to be difficult or even impossible unless the respective MbtH-like protein was coexpressed simultaneously (6 -8).…”

“…If the adenylating enzyme CmnO and the MbtH-like protein CmnN were purified separately, a mixture of both in a 1:1 molar ratio showed only low activity. 10-Fold higher activity was observed when the MbtH-like protein was added in a 16 -32-fold molar excess (6). Therefore, the composition of the complex of MbtH-like proteins with adenylating enzymes is still obscure.…”

Assessing the progress of Mycobacterium tuberculosis H37Rv structural genomics

“…Siroheme-dependent sulfite reductase (Rv2391) was hence solubly produced by coproducing uroporphyrinogen III C-methyl-transferase (Rv2847c) (56). Similarly, nonribosomal peptide synthetases, MbtE and MbtF of mycobactin biosynthesis, were solubly produced only by co-producing 8 kDa MbtH with a previously unknown role in the pathway (57,58). Yet another strategy of obtaining structural data for difficult or insoluble Mtb proteins is to replace them with mycobacterial orthologues.…”