Structural Basis of the Interaction of MbtH-like Proteins, Putative Regulators of Nonribosomal Peptide Biosynthesis, with Adenylating Enzymes

Abstract

Background: MbtH-like proteins are required for many adenylation reactions in nonribosomal peptide biosynthesis. Results: We present the crystal structure of the adenylating enzyme SlgN1, involved in the biosynthesis of the antibiotic streptolydigin, and analyze its interface with an MbtH-like domain. Conclusion: and Ala-433 (adenylation domain) are required for domain interaction. Significance: MbtH-like proteins activate adenylating enzymes but make no direct contact with their substrates.

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

“…The VinNN structure has a five-layered ␣␤␣␤␣ sandwich fold as observed in other crystal structures of adenylation enzymes (2, 26, 38 -41) (Fig. 2A) A structural comparison of VinNN with other adenylatebound adenylation enzymes, including PheA and SlgN1 (2,26,35,41,43), showed that VinNN has a similar cleft for adenylate binding on the surface of the N-terminal domain. VinN has several conserved residues at that cleft (Fig.…”

“…Crystallization of these enzymes containing flexible C-terminal parts is challenging. Hence, there are some examples of crystal structures in which only the N-terminal part has been used for crystallization (26,38,39). Therefore, we constructed the heterologous expression system of the VinNN protein containing only the N-terminal domain (Met 1 -Arg 426 ) and then attempted to crystallize the VinNN protein.…”

“…As VinN exhibits relatively low sequence identity with well studied adenylation enzymes such as the NRPS adenylation domain, the substrate specificity of VinN cannot be predicted from the specificityconferring code. The crystal structure of SlgN1, which catalyzes the adenylation of 3-MeAsp in the biosynthesis of streptolydigin, has been reported recently (26). SlgN1 recognizes (2S,3S)-3-MeAsp as an ␣-amino acid substrate and adenylates the carboxyl group at the C1 position (Fig.…”

“…5). The SlgN1 structure suggests that this polar Gln 305 residue might be involved in carboxyl group recognition, although the structure of the complex with 3-MeAsp was not reported (26) (Fig. 4D).…”

An environmental bacterial taxon with a large and distinct metabolic repertoire

“…The PCR products were ligated into the vectors pGEM-T Easy or pBluescript SK(1) and transformed into E. coli DH5a for subcloning into pET28b. Based on recent findings that many A domains are only active in vitro when coexpressed with a MbtH-like protein 60,61 , each of the Histagged adenylation domains were coexpressed with KerL, a MbtH-like protein from the ker pathway. KerL was cloned into the co-expression vector pCDF-DUET.…”