Biosynthesis of Antituberculosis Antibiotic Capreomycin Involves a trans-Iterative Adenylation Domain within the Nonribosomal Peptide Synthetase Machinery

Abstract

Capreomycin (CMN) is a nonribosomal peptide (NRP) antituberculosis antibiotic. CMN biosynthesis involves a non-canonical trans-iterative adenylation (A) domain. Here, we report that the A domain-less nonribosomal peptide synthetase (NRPS) module CmnI utilizes another module's A domain CmnA-A₁ to load the required amino acid onto its thiolation (T) domain. This study provides evidence of an unusual mode of NRP biosynthesis in bacteria, involving a trans-iterative A domain in the NRPS machinery.

1

Biosynthesis of CMN and VIO: (a) Structures of the four CMN derivatives and VIO and (b) composition and arrangement of the NRPSs of CMN and VIO. In both CMN and VIO, NRPS module 3 lacks an A domain. l-Cap and β-Uda represent l-capreomycidine and β-ureidodehydroalanine, respectively. The red dashed arrows indicate that CmnA-A₁ and VioA-A₂ load amino acids across modules onto CmnI-T and VioI-T, respectively.

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In vivo enzymatic reactions and mass analysis of CmnI. Deconvoluted protein mass spectra of purified CmnI: (a) apo-form CmnI (expression of only cmnI in E. coli), (b) holo-form CmnI (expression of cmnI and svp in E. coli), and (c and d) CmnI loaded with l-Dap (co-expression of cmnI, svp, cmnA-A ₁ , and cmnN in E. coli). Spectra c and d represent the conditions without and with l-Dap added to the culture medium, respectively. The calculated molecular weight of apo-form CmnI is 61 484.72 Da.

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In vitro enzymatic reactions and mass analysis of CmnI-T: (a) apo-form CmnI-T, (b) holo-form CmnI-T, and (c) CmnI-T loaded with l-Dap. In the reaction of spectrum c, CmnI-T, CmnA-A₁–CmnN, as well as the substrate l-Dap and the cofactors ATP and Mg²⁺ were added. Subsequently, CmnI-T was purified for an intact protein mass analysis. The calculated molecular weight of apo-form CmnI-T is 12 316.88 Da.

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Bioinformatics analysis of the CmnI homologues: (a) 987 CmnI homologues classified by the SSN analysis at an e value of 10^–80, with CmnI and the 12 homologues forming a distinct group in the SSN, highlighted in red, and (b) biosynthetic gene clusters identified by antiSMASH containing 12 CmnI homologues highlighted in panel a. The proposed biosynthetic functions of the open reading frames (ORFs) are indicated below the figure.

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Structure of CmnI: (a) crystal structure of the C domain of CmnI (CmnI-C), with the N- and C-terminal subdomains colored deep blue and light blue, respectively, the floor loop and bridging region colored yellow and orange, respectively, and the C domain conserved motif HHXXXD colored green, (b) local view of the putative CmnI-C active-site cavity, with the second histidine, His249, of the conserved motif shown as green sticks and PEG shown as black sticks, and (c) AlphaFold models of the T domain of CmnI (CmnI-T), with the N-terminal region of CmnI-T predicted to be a disorder region, the five predicted models of CmnI-T shown in five different colors, and the conserved T domain serine, Ser56, shown as sticks.