title: Structural basis of the interaction of MbtH-like proteins, putative regulators of nonribosomal peptide biosynthesis, with adenylating enzymes.
abstract: The biosynthesis of nonribosomally formed peptides (NRPs), which include important antibiotics such as vancomycin, requires the activation of amino acids through adenylate formation. The biosynthetic gene clusters of NRPs frequently contain genes for small, so-called MbtH-like proteins. Recently, it was discovered that these MbtH-like proteins are required for some of the adenylation reactions in NRP biosynthesis, but the mechanism of their interaction with the adenylating enzymes has remained unknown. In this study, we determined the structure of SlgN1, a 3-methylaspartate-adenylating enzyme involved in the biosynthesis of the hybrid polyketide/NRP antibiotic streptolydigin. SlgN1 contains an MbtH-like domain at its N terminus, and our analysis defines the parameters required for an interaction between MbtH-like domains and an adenylating enzyme. Highly conserved tryptophan residues of the MbtH-like domain critically contribute to this interaction. Trp-25 and Trp-35 form a cleft on the surface of the MbtH-like domain, which accommodates the alanine side chain of Ala-433 of the adenylating domain. Mutation of Ala-433 to glutamate abolished the activity of SlgN1. Mutation of Ser-23 of the MbtH-like domain to tyrosine resulted in strongly reduced activity. However, the activity of this S23Y mutant could be completely restored by addition of the intact MbtH-like protein CloY from another organism. This suggests that the interface found in the structure of SlgN1 is the genuine interface between MbtH-like proteins and adenylating enzymes.
authors.affiliation: Interfakultäres Institut für Biochemie, Eberhard Karls-Universität Tübingen, 72076 Tübingen, Germany.
authors.forename: Dominik A
authors.initials: DA
authors.lastname: Herbst
authors.forename: Björn
authors.initials: B
authors.lastname: Boll
authors.forename: Georg
authors.initials: G
authors.lastname: Zocher
authors.forename: Thilo
authors.initials: T
authors.lastname: Stehle
authors.forename: Lutz
authors.initials: L
authors.lastname: Heide
mesh_terms: D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000096:biosynthesis* / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D046908:Peptide Biosynthesis, Nucleic Acid-Independent / Q000235:genetics*; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D000249:Adenosine Monophosphate; C100307:streptolydigin; D014364:Tryptophan; D010453:Peptide Synthases; D000409:Alanine