Stuffed Methyltransferase Catalyzes the Penultimate Step of Pyochelin Biosynthesis

Abstract

Nonribosomal peptide synthetases use tailoring domains to incorporate chemical diversity into the final natural product. A structurally unique set of tailoring domains are found to be stuffed within adenylation domains and have only recently begun to be characterized. PchF is the NRPS termination module in pyochelin biosynthesis and includes a stuffed methyltransferase domain responsible for S-adenosylmethionine (AdoMet) dependent N-methylation. Recent studies of stuffed methyltransferase domains propose a model in which methylation occurs on amino acids after adenylation and thiolation rather than after condensation to the nascent peptide chain. Herein, we characterize the adenylation and stuffed methyltransferase didomain of PchF through the synthesis and use of substrate analogs, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an S N 2 reaction after thiolation, condensation, cyclization and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

An overview of siderophore biosynthesis among fluorescent Pseudomonads and new insights into their complex cellular organization

“…A second molecule of cysteine is coupled to Dha by another NRPS enzyme, PchF, again under the control of PchC (Reimmann et al, 2004). This second cysteine undergoes cyclisation by the cycling module of PchF, to form nor-pyochelin, and methylation on the second thiazolidine cycle by the methylation module of PchF (Patel et al, 2003;Ronnebaum et al, 2019). The synthesized PCH is then released by the reductase PchG Reimmann et al, 2001).…”

Deciphering Biosynthetic Enzymes Leading to 4-Chloro-6-Methyl-5,7-Dihydroxyphenylglycine, a Non-Proteinogenic Amino Acid in Totopotensamides

“…Two C -methyltransferases AcmI and AcmL were shown to utilize free amino acids as substrates in the biosynthesis of the actinomycin chromophore (Figure B) . Recently, the C -methyltransferases MdpB1 and PokMT1 from maduropeptin and polyketomycin biosynthetic pathways, respectively, were established to catalyze C -methylations on CoA-activated aromatic substrates (Figure B). , Interestingly, a growing number of methylation domains were found to be embedded within A domains of NRPS proteins to catalyze N -, O -, or S -methylations of T domain-tethered amino acids. − A phylogenetic analysis shows that TotM belongs to a unique clade of methyltransferase-domain containing proteins (Figure B), which are proposed to use T domain-bound amino acids as substrates. Similarly, the methyltransferase Orf23 for the C -methylation of DPG in ristocetin (Figure ) falls into the same clade (Figure B).…”

Characterization of a Unique Interrupted Adenylation Domain That Can Catalyze Three Reactions

“…For example, for the uninterrupted A domains PheA, 7 EntF with YbdZ, 30 and Ecm6(A 1 ) with Ecm8 11 (note that YbdZ and Ecm8 are MbtH-like protein partners for those corresponding A domains), k cat values were 62, 57 ± 6, and 5.8 ± 0.6 min −1 , respectively, and K m values were 0.9, 0.149 ± 0.058, and 1.5 ± 0.4 mM, respectively. For previously published activity of native interrupted A domains, TioS(A 3a M 3S A 3b ) with TioT 9 and PchF with PA2412 31 (note that TioT and PA2412 are MbtH-like protein partners for those corresponding A domains), k cat values of 7.72 ± 0.03 and 3.2 ± 0.2 min −1 as well as K m values of 0.140 ± 0.003 and 0.041 ± 0.008 mM were reported, respectively (note that the activity of the PchF A domain was measured by a continuous kinetic assay by using inorganic pyrophosphatase, 32 in line with those observed for ColG(AM s M b A)).…”