Deciphering Nature’s Intricate Way of N,S-Dimethylating l-Cysteine: Sequential Action of Two Bifunctional Adenylation Domains

Abstract

Dimethylation of amino acids consists of an interesting and puzzling series of events that could be achieved, during nonribosomal peptide biosynthesis, either by a single adenylation (A) domain interrupted by a methyltransferase (M) domain or by the sequential action of two of such independent enzymes. Herein, to establish the method by which Nature N,S-dimethylates l-Cys, we studied its formation during thiochondrilline A biosynthesis by evaluating TioS(AMAT) and TioN(AMA). This study not only led to identification of the exact pathway followed in Nature by these two enzymes for N,S-dimethylation of l-Cys, but also revealed that a single interrupted A domain can N,N-dimethylate amino acids, a novel phenomenon in the nonribosomal peptide field. These findings offer important and useful insights for the development and engineering of novel interrupted A domain enzymes to serve, in the future, as tools for combinatorial biosynthesis.

Synthesis, Characterisation and Reactivity of 3‐Mercaptopyruvic Acid

“…Although the use of the dimer is, under most circumstances, not problematic for chemical, biochemical or biological studies (see below), several major drawbacks are associated with this synthesis: 1) a tedious and harmful purification of sodium hydrogen sulfide is required prior to the reaction to avoid erratic yield and purity; and 2) a second purification step is necessary prior to certain biochemical experiments to remove traces of hydrosulfide, which could affect the data . Last year, an alternative synthesis was proposed, but the resulting product was barely characterised. Herein, we present a synthetic route that unambiguously leads to 3‐mercaptopyruvic acid (hereafter, we use 3‐MPH to refer to 3‐mercaptopyruvic acid synthesised in this work, and 3‐MP for commercially available “sodium 3‐mercaptopyruvate” or for the product derived from the synthesis with sodium hydrosulfide and bromopyruvic acid, regardless of the protonation state of the compounds in solution) with purity greater than 97 %, after simple purification steps.…”

Lessons learned in engineering interrupted adenylation domains when attempting to create trifunctional enzymes from three independent monofunctional ones

“…Aer verifying that the [methyl-3 H]SAM stock had not degraded as a result of long-term storage and/or multiple freeze-thaw cycles, we thought perhaps the issue lied in the ability of the engineered interrupted A domains to activate and load L-Ser onto the T domain, for if the T domain cannot be loaded, methylation cannot take place. 9 Therefore, we supplemented the reactions with wt Ecm6(A 1 ), which is able to load the T domain with L-Ser, 12 however, we were still unable to detect increasing methylation compared to the no substrate negative controls. To assess if perhaps loading of the T domain was the issue, we also investigated the engineered interrupted A domain's ability to load L-Ser onto Ecm6(T 1 ) by HPLC.…”

Refining and expanding nonribosomal peptide synthetase function and mechanism

“…Modules 3 and 4 make up a two module-containing NRPS, TioS. Similar to the A domain of KtzH, both of the A domains in TioS are embedded with M domains between the a8 and a9 motif, suggesting that one or both are involved in the incorporation of N-methyl-L-Cys [72]. A discrete and apparently extra A domain protein called TioN is also encoded within the gene cluster [1].…”

“…Subsequently, TioN was shown to catalyze methylation of L-Cys-S-CP, and although it was believed to be S-methylation, the regiochemistry of methylation was not determined. Following the functional assignment of TioN, the role of TioS was investigated in closer detail [72]. The thiocoraline gene cluster also produces thiochondrilline A, which is a pentapeptide that is, in essence, half of the thiocoraline dimer with a few structural variations ( Figure 8A) [116].…”