How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic.
The 39-kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a 'Trojan horse' antibiotic. The approximately 260-residue C-terminal region of MccB is homologous to ubiquitin-like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB-catalysed C-terminal MccA-acyl-adenylation is reminiscent of the E1-catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C-terminal di-glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C-terminal Asn. Furthermore, after an intramolecular rearrangement of MccA-acyl-adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate-linked analogue of acyl-adenylated aspartic acid. We report six-crystal structures of MccB in apo, substrate-, intermediate-, and inhibitor-bound forms. Structural and kinetic analyses reveal a novel-peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic-active site for catalysing dual adenosine triphosphate-consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two-step activation for generating the peptidyl-antibiotic MccC7.
Regni
Catherine A
CA
Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Roush
Rebecca F
RF
Miller
Darcie J
DJ
Nourse
Amanda
A
Walsh
Christopher T
CT
Schulman
Brenda A
BA
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D001430:Bacteriocins / Q000096:biosynthesis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D029968:Escherichia coli Proteins; D009711:Nucleotides; D010455:Peptides; C020411:microcin; D001224:Aspartic Acid; D008025:Ligases; C542496:MccB protein, E coli; D044764:Ubiquitin-Activating Enzymes