Also, optional or modifying domains can be present in one or more modules, such as methyltransferase (De Mattos-Shipley et al., 2018), epimerization (Samel et al., 2014), formylation (Schoenafinger et al., 2006), and oxidase (Schneider et al., 2003) domains. The domains A, PCP, and C/Cy are strictly required for NRP synthesis, and therefore, a minimal module contains all three of them (initiation modules can lack a C domain as they represent the starter units of NRPS). As a result, the latter is released from the ppant moiety, and the newly synthesized intermediate is now ready to be transported to the next condensation domain (Condurso & Bruner, 2012 Koglin & Walsh, 2009 Marahiel et al., 2009 Reimer et al., 2018 Schwarzer et al., 2003 Strieker et al., 2010 Walsh, 2016). Here, peptide formation occurs via nucleophilic attack of the α-amino group of the downstream substrate to the activated carboxy group of the upstream substrate. The loaded substrates are subsequently transported to the active site of the condensation (C) domain (or a cyclization domain, Cy).
The ppant is a CoA (Coenzyme A)-derived cofactor, covalently attached to a highly conserved residue of serine of the T domain. The activated substrate is then transferred via a thioesterification reaction to the thiolation (T)-or peptide carrier protein (PCP)-domain through the phosphopantetheine (ppant) arm, with AMP being released. In this process, ATP is consumed and inorganic pyrophosphate (PP i) is released. NRP synthesis generally starts in every module at the adenylation (A) domain, which recognizes a specific substrate and catalyzes its activation as (amino) acyl-AMP. The first enzyme of the pathway, ACVS, belongs to the family of nonribosomal peptide synthetases (NRPS), multimodular enzymes that synthesize small peptides (NRP) by a mechanism that does not require messenger RNA, unlike ribosomal protein synthesis (Marahiel et al., 2009 Reimer et al., 2018). The second step requires the enzyme isopenicillin N synthase (IPNS, formerly also ACV cyclase), which catalyzes the formation of the β-lactam ring. First, the l-δ-(α-aminoadipoyl)- l-cysteinyl- d-valine synthetase (ACVS) provides the linear tripeptide (L,L,D)-ACV, a precursor of all β-lactam antibiotics. Two distinct enzymatic steps are involved in the production of the β-lactam ring. The biosynthetic pathway of penicillin (and other β-lactam antibiotics such as cephalosporins and cephamycins) has been extensively characterized in the past decades, resulting in detailed knowledge on the key enzymes involved and the mechanistic aspects of its biosynthesis (Baldwin & Abraham, 1988 Coque et al., 1991 Liras, 1999 Martín, 1998 Martín et al., 1999 Ozcengiz & Demain, 2013 Queener, 1990 Roach et al., 1997 Schofield et al., 1997 Tahlan et al., 2016 Wu et al., 2012).
Since then, many more classes of compounds have been identified or synthesized, but penicillin and its (semi-) synthetic derivatives are still among the most common antibiotics used worldwide. The discovery of penicillin has been a crucial turning point in human history, completely revolutionizing the treatment of bacterial infections. The data indicate that the N-terminal domain is important for catalysis. Deletion variants of both enzymes were generated to investigate the potential impact on penicillin biosynthesis in vivo and in vitro. Bioinformatic analyses revealed the presence of a previously unidentified domain at the N-terminus which is structurally related to condensation domains, but smaller in size. Here we carried out an in-depth investigation on the architecture of the first module of the ACVS enzymes from the fungus Penicillium rubens and the bacterium Nocardia lactamdurans. The first module is highly specific and is the only NRPS unit known to recruit and activate the substrate l-α-aminoadipic acid, which is coupled to the α-amino group of l-cysteine through an unusual peptide bond, involving its δ-carboxyl group. The enzyme has been extensively characterized in terms of tripeptide formation and substrate specificity. The l-δ-(α-aminoadipoyl)- l-cysteinyl- d-valine synthetase (ACVS) is a trimodular nonribosomal peptide synthetase (NRPS) that provides the peptide precursor for the synthesis of β-lactams.
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