Loading module

L, loading module DH, dehydratase KS, p-ketosynthase KR, ketoreductase MT methyltransferase PS, pyran synthase DHh and KRh are DH and KR-like sequences, together with the FkbH domain, they are involved in the formation of D-lactate starter unit HMG-CS, hydroxy-methyl-glutaryl CoA synthase. Acyl-carrier-protein domains are shown as small filled balls with chain attached by the thiol group. The box shows the HMG-CS pathway for the formation of exocyclic enoate. 

PKS loading module 1 module 2 module 3 module 4 release... 

Loading Module 1 Module 2 Module3 Module 4 Module5 Modules Cy°li2ation... 

Most of the latest publications on NRPS substrate specificity are focused on A domain specificity because their substrate screening is straightforward in terms of biosynthetic substrate form (free amino acids/fatty acids/aryl acids) and T domain substrates (one T domain). Four studies focus on substrate specificity of NRPS loading modules of microcystin biosynthesis,97 mycosubtilin biosynthesis,51 daptomycin biosynthesis,108 and leinamycin biosynthesis.108 The A domains of microcystin, mycosubtilin, and daptomycin biosynthesis initiation showed fatty acid specificity. The initial domain from leinamycin biosynthesis has D-amino acid specificity. Another paper presents the elucidation of aryl acid-specific AsbC adenylation enzyme from petrobactin biosynthesis.104... 

The first example in which MS was utilized to determine the specificity of an A domain from an NRPS gene cluster was published in a joint effort by the Moore and Kelleher laboratories. Hicks et al. 97 investigated the substrate specificity of the loading module McyG of microcystin synthetase. Microcystin is a cyclic NRPS-PKS hybrid toxin derived from various cyanobacteria genera. The initiation module McyG comprises an A-T didomain (Figure 14(b)), which was predicted based on the 10 letter code to activate and load... 

Figure 14 In vivo and in vitro substrate screening of loading module of microcystin biosynthesis, (a) Microcystin and Adda, (b) Loading protein McyG of microcystin synthetase, (c) In vitro and in vivo substrate screening assays of McyG AT. (d) Characterized substrates of McyG AT, ,Vo and McyG AT, vitm by ESI-FTMS (observed and calculated mass shifts from holo McyG AT active site).

Figure 17 The loading module of leinamycin biosynthesis, (a) Loading module components (LnmQ and LnmP) of leinamycin synthetase and leinamycin structure, (b) Substrate screening assays of LnmQ. D-Alanine and glycine loading was detected by ESI-MS (observed and calculated mass shifts of holo LnmP).

The recent studies of Hansen et al51 and Wittmann et al,109 revealed a new mechanism for lipidation of lipopeptide biosynthesis such as mycosubtilin or daptomycin biosynthesis by application of ESI-FTMS. Both papers describe that fatty acid incorporation is catalyzed by an A domain with fatty acid specificity in the loading module of the mycosubtilin NRPS (Figure 15) or by a preassembly line A and T domain in daptomycin biosynthesis (Figure 16). 

The avermectin PKS loading module has been used to generate a hybrid PKS system by replacing the loading module of DEBS 1-TE. The new hybrid PKS produced new hybrid polyketide (triketide lactones) which incorporated the isobutyrate and 2-methylbutyrate starter acids of avermectin biosynthesis, as well as the normal acetate and propionate starter units of erythromycin biosynthesis [51]. 

The loading module comprises three domains. The first (CL) shows homology to ATP-dependent carboxylic acid-CoA ligases, the second is a putative enoyl reductase (ER) and the third an ACP. The probable sequence of operations starts with the enoic acid 74 derived from shikimic acid which is reduced by the ER domain. The first domain will activate the carboxylic acid to an active acyl derivative ready for transfer to the thiol residue of the ACP. The final saturated product will end up attached to the ACP as a thioester derivative ready for transfer to the KS domain of the first chain extension module. The timing of the reduction in this sequence of operations cannot be predicted. 

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