Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S-Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases

Abstract

Identifying an enzyme's substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.

Identifying Unknown Enzyme–Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry

“…In this case, the isotopically labeled probe was not used, resulting in a single peak. Isotopically labeled spectra were previously reported …”

“…In transmethylation, the enzyme transfers a methyl from common donor AdoMet to a nucleophilic substrate, such as thiopurine methyltransferase (TPMT, EC 2.1.1.67) which methylates thiophenols (Scheme A). For this system, the selected IsoLAIT probe was S ‐adenosyl‐ l ‐vinthionine (AdoVin) …”

“…We posited that TPMT would catalyze the formation of a substrate–probe adduct (Scheme B); indeed adduct formation between the substrate's thiol and the vinyl sulfonium in AdoVin was confirmed . However, it was not clear whether such an [enzyme ⋅ substrate ⋅ probe] complex would bind sufficiently to survive native MS. To investigate, we prepared both in vitro and ex vivo (i.e., Escherichia coli cell lysate) samples (Figure S1 in the Supporting Information).…”

“…Preparation of S ‐adenosylvinthionine (AdoVin) : S ‐Adenosyl‐ l ‐vinthionine [CAS 83 768‐89‐2] was prepared enzymatically by using S ‐adenosyl‐methionine synthetase (MAT, EC 2.5.1.6) with both labeled (+15 Da) and unlabeled ATP (10 m m ), along with vinthionine (1 m m ). The reaction proceeded in a potassium phosphate buffer (50 m m , pH 8.0) with KCl (5 m m ), and MgCl 2 (2.5 m m) and was initiated with MAT (50 μ m ) and incubated at 37 °C.…”

“…We posited that TPMT would catalyze the formation of a substrate-probe adduct [7, 24–29] (Scheme 1B); indeed adduct formation between the substrate’s thiol and the vinyl sulfonium in AdoVin was confirmed. [22] However, it was not clear whether such an [enzyme•substrate-probe] complex would bind sufficiently to survive native MS. To investigate, we prepared both in vitro and ex vivo (i.e., E. coli cell lysate) samples (Figure S1). AdoVin was prepared enzymatically [22] with S -adenosyl-methionine synthetase (MAT, EC 2.5.1.6) using both labeled (+15 Da) and unlabeled ATP along with vinthionine (Figure S1).…”