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 analogues, steady-state kinetics, and onium chalcogen effects. We provide evidence that methylation occurs through an S_N2 reaction after thiolation, condensation, cyclization, and reduction of the module substrate cysteine and is the penultimate step in pyochelin biosynthesis.

Figure 1:. Synthesis of pyochelin.

A) Biosynthetic pathway for pyochelin production. PchA, an isochorismate synthase, and PchB, an isochorismate pyruvate lyase, convert chorismate to salicylate. PchD-G consists of 12 different domains constituting three NRPS modules with three tailoring domains. Together, they assemble pyochelin from salicylate and two l-cysteines, with co-substrates AdoMet, ATP, and NADPH. AdoMet = S-adenosylmethionine, AdoHcys = S-adenosylhomocysteine. PchD is a stand-alone adenylation domain (A; blue). PchE has N- and C-terminal thiolation domains (T; yellow), a cyclization domain (Cy; grey), an adenylation domain (A; blue) and a stuffed epimerase domain (E; purple). PchG is a stand-alone NADPH-dependent reductase. PchF is a full-NRPS module consisting of 5 different domains: a cyclization domain (Cy; grey), adenylation domain (A; blue), stuffed methyltransferase tailoring domain (M; red), thiolation domain (T; yellow), and thioesterase domain (TE; green). B) PchF-FL refers to the “full-length” protein, whereas PchF-AMT only includes the stuffed adenylation methyltransferase didomain and the thiolation domain. G667I-PchF-AMT has a mutation to the proposed AdoMet binding domain of the variant PchF-AMT. C) The proposed order of chemistries performed by PchF. PchF incorporates l-cysteine to the growing peptide chain. The Cy-domain of PchF continues chain elongation by generating a peptide bond between l-cysteine and the upstream hydroxyphenyl-d-thiazoline moiety of PchE (an intermediate attached to the Ppant of the T-domain of PchE). The Cy-domain then cyclizes the cysteine to form a hydroxyphenyl-(d)-thiazoline-(l)-thiazoline intermediate. PchG (green) reduces the terminal thiazoline of the hydroxyphenyl-bis-heterocycle to thiazolidine using NADPH. The stuffed methyltransferase domain of PchF catalyzes the AdoMet-dependent methyl transfer (highlighted in red) onto the nitrogen of the newly reduced thiazolidine ring before transferring the complete heterocyclic siderophore to the TE-domain releasing the fully mature pyochelin by hydrolysis in the TE-domain. The alternate synthetic schemes where methyl transfer occurs at a different position in the sequence of biosynthesis are shown in Figure S1. D) During reconstitution assays, premature hydrolysis releases peptidyl intermediates during biosynthesis. Intermediates previously recovered by Walsh and colleagues are noted with an asterisk. Intermediate analogs used as potential substrates in this study are surrounded by a square. Nomenclature of intermediates is as follows: HP, hydroxyphenyl; T_ox, thiazole; T, thiazoline; T_red, thiazolidine; CO₂- carboxyl; Et, ethyl; Me/M, methyl.

Figure 2:. Adenylation activity of PchF variants.

PchF catalyzes the adenylation of l-cysteine by ATP forming an aminoacyl-adenylate bond, releasing pyrophosphate. In the adenylation assay (Figure S3), pyrophosphate is converted to two inorganic phosphates by inorganic pyrophosphatase. 7-methylguanosine is then phosphorylated by purine nucleoside phosphorylase to generate 7-methylguanine, which absorbs at 360 nm. A) PchF variants do not turnover with l-cysteine and ATP (A), but do with the addition of hydroxylamine as a nucleophilic surrogate (B). Michaelis-Menten steady-state kinetics (C) were performed to compare the l-cysteine adenylation activity of PchF-FL, PchF-AMT, and the methyltransferase null mutant of G667I-PchF-AMT.

Figure 3:. Methylated Product Formation Assay.

PchF catalyzes methyl transfer from its natural co-substrate AdoMet to the synthesized substrate analog, HPT_oxT_red-CO₂Et. Steady-state Michaelis-Menten kinetics were performed by a discontinuous assay. Substrate analog (eluting at 20 min) and product analog (eluting at 21.6 min) were separated by a C18 column using HPLC.

Figure 4:. S-adenosylhomocysteine Formation Assay.

An orthogonal assay was used to determine if l-Cys-OEt was a substrate analog of PchF methyl transfer by detecting the formation of coproduct, AdoHCys, by UPLC separation. PchF-FL or PchF-AMT was incubated with 1 mM AdoMet and either no substrate, 30 μM HPT_oxT_red-CO₂Et (blue), or 30 μM or 3 mM l-Cys-OEt for 3 hours before being separated by UPLC. A) A bar graph showing the peak area quantitation of AdoHCys for reactions with 1) 30 μM HPT_oxT_red-CO₂Et but without enzyme; 2) 1 μM PchF-AMT without substrate; 3) 1 μM PchF-FL without substrate; 4) 1 μM PchF-AMT with 30 μM HPT_oxT_red-CO₂Et; 5) 1 μM PchF-FL with 30μM HPT_oxT_red-CO₂Et; 6) 1 μM PchF-AMT with 30 μM l-Cys-OEt; 7) 1 μM PchF-FL with 30 μM l-Cys-OEt; 8) 1 μM PchF-AMT with 3 mM l-Cys-OEt; and 9) 1 μM PchF-FL with 3mM l-Cys-OEt. Comparative UPLC traces for reactions using B) PchF-FL and C) PchF-AMT are displayed when using no substrate (black), 3mM l-Cys-OEt (red), and 30 μM HPT_oxT_red-CO₂Et (blue). AdoHCys formation is within error of negative controls when l-Cys-OEt is used as a substrate. In contrast, equimolar amounts of AdoHCys are generated when HPT_oxT_red-CO₂Et is used as a substrate analog.

Scheme 1:. Substrate analog synthesis of HPT_oxT-CO₂⁻ HPT_oxT-CO₂Et, HPT_oxT_red-CO₂⁻HPT_oxT_red-CO₂Et and HPT-Cys-Me.

Substrate analogs were generated to assess the methyltransferase capabilities of PchF. Reagents and conditions: a) Pd(PPh₃)₄, K₂CO₃/DME, H₂O, 150 °C, 49%; b) MeOH, K₂HPO₄(aq) (pH 6.5), 60 °C, 70%; c) SOCl₂/EtOH, 40 °C, 49%; d) Pd(PPh₃)₄, K₂CO₃/DME, H₂O, 100°C, 70%; e) EtOH/KCO₂CH₃ (aq), RT, 95%; f) SOCl₂/EtOH, 40°C, 55%; g) DIEA, COMU/DMF, 0°C 1hr, RT 3hr, 12%; h) generated in the presence of 5mM TCEP in vitro.