Reprogramming Nonribosomal Peptide Synthetases for “Clickable” Amino Acids

Abstract

Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes that produce a wide array of bioactive peptides. Here we show that a single tryptophan-to-serine mutation in phenylalanine-specific NRPS adenylation domains enables the efficient activation of non-natural aromatic amino acids functionalized with azide and alkyne groups. The resulting 10(5)-fold switch in substrate specificity was achieved without appreciable loss of catalytic efficiency. Moreover, the effective communication of the modified A domains with downstream modules in dipeptide synthetases permitted incorporation of O-propargyl-L-tyrosine into diketopiperazines both in vitro and in vivo, even in the presence of competing phenylalanine. Because azides and alkynes readily undergo bioorthogonal click reactions, reprogramming NRPSs to accept non-natural amino acids that contain these groups provides a potentially powerful means of isolating, labeling, and modifying biologically active peptides.

A Subdomain Swap Strategy for Reengineering Nonribosomal Peptides

“…We speculate that binding pocket mutagenesis will be the preferred instrument to achieve small structural changes in substrate preference Evans et al, 2011;Kries et al, 2014;Thirlway et al, 2012) since this approach is least likely to wreak havoc on the structural integrity of the synthetase. However, second-shell and long-range interactions that may be crucial for larger changes in specificity are ignored by this approach.…”

“…The expected product, D-Val-L-Pro DKP, was detected by LC-MS along with 25% unepimerized L-Val-L-Pro DKP ( Figure 4B). The engineered sdV-GrsA/GrsB1 synthetase incorporated L-Val with a k obs of 0.003 min À1 (Figure 4C), 300 times slower than the wild-type GrsA/GrsB1 system, which produces D-Phe-L-Pro DKP with a k obs of 0.9 min À1 (Kries et al, 2014). Despite the large structural transition from the native substrate L-Phe to L-Val, all domains remain at least partially functional.…”

“…GrsB1 is a typical proline-specific elongation module with C, A, and T domains. DKP synthetases provide a convenient sensor for peptide formation activity because the required modules are reasonably small and can be expressed and assayed in vitro (Gruenewald et al, 2004;Kries et al, 2014;Stachelhaus et al, 1998). The cyclized DKP products are readily detected by liquid chromatography (LC)-MS.…”

“…Construction of plasmids pMG211_mgrsAA 0 , pSU18_mgrsA, and pTrc99a_ grsB1 has been described previously (Kries et al, 2014). In plasmids pMG211_mgrsAA 0 and pSU18_mgrsA, the subdomain stretch of grsA A is flanked by restriction sites that can be harnessed for exchanging the subdomain.…”

“…As a consequence, domain and module swaps often result in low activities (Fischbach et al, 2007;Schneider et al, 1998;Stachelhaus et al, 1995). Alternatively, synthetases can be tailored by fine-tuning individual domains, usually the specificity-determining A domains Evans et al, 2011;Kries et al, 2014;Thirlway et al, 2012), but the specificity changes that can be achieved are typically conservative.…”