BlmIII and BlmIV nonribosomal peptide synthetase-catalyzed biosynthesis of the bleomycin bithiazole moiety involving both in cis and in trans aminoacylation.
Cloning and sequence analysis of the bleomycin (BLM) biosynthetic gene cluster predicted that the two nonribosomal peptide synthetases (NRPSs), BlmIV and BlmIII, are responsible for the biosynthesis of the BLM bithiazole moiety. BlmIV is a seven domain (C(2)-A(2)-PCP(2)-Cy(1)-A(1)-PCP(1)-Cy(0)) NRPS, and BlmIII is a three domain (A(0)-PCP(0)-Ox) NRPS. The three domains of Cy(1)-A(1)-PCP(1) residing on the BlmIV subunit, the four domains of Cy(0) residing on the BlmIV subunit, and A(0)-PCP(0)-Ox residing on the BlmIII subunit constitute the two thiazole-forming NRPS-1 and NRPS-0 modules, respectively. BlmIII-A(0) was predicted to be nonfunctional, raising the question of how the NRPS-0 module activates and loads the Cys substrate to its cognate BlmIII-PCP(0). The NRPS-0 module consists of domains residing on two different subunits, requiring precise protein-protein interaction. Here, we report the production of the BlmIV and BlmIII NRPSs as an excised domain(s), module, or intact subunit form and biochemical characterizations of the resultant enzymes in vitro for their roles in BLM bithiazole biosynthesis. Our results (a) confirm that BlmIII-A(0) is a naturally occurring nonfunctional mutant, (b) demonstrate that BlmIV-A(1) activates Cys and catalyzes both in cis aminoacylation of BlmIV-PCP(1) (for NRPS-1) and in trans aminoacylation of BlmIII-PCP(0) (for NRPS-0), and (c) reveal that the C-terminus of the BlmIV subunit, characterized by the unprecedented AGHDDD(G) and PGHDDG repeats, is absolutely required for in trans aminoacylation of BlmIII-PCP(0). These findings underscore the flexibility and versatility of NRPSs in both structure and mechanism for natural product biosynthesis and provide an outstanding opportunity to study the molecular recognition and protein-protein interaction mechanism in NRPS assembly line enzymology.
Du
Liangcheng
L
Division of Pharmaceutical Sciences and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705, USA.
Chen
Mei
M
Zhang
Yang
Y
Shen
Ben
B
D000215:Acylation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001761:Bleomycin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003545:Cysteine / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D013237:Stereoisomerism; D013844:Thiazoles / Q000737:chemistry* / Q000378:metabolism*
D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D013844:Thiazoles; D001761:Bleomycin; D010453:Peptide Synthases; D003545:Cysteine