Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an α,β-unsaturated thioester

Abstract

Thioesterases catalyze hydrolysis of acyl thioesters to release carboxylic acid or macrocyclization to produce the corresponding macrocycle in the biosynthesis of fatty acids, polyketides, or nonribosomal peptides. Recently, we reported that the thioesterase CmiS1 from sp. MJ635-86F5 catalyzes the Michael addition of glycine to an α,β-unsaturated fatty acyl thioester followed by thioester hydrolysis in the biosynthesis of the macrolactamantibiotic cremimycin. However, the molecular mechanisms of CmiS1-catalyzed reactions are unclear. Here, we report on the functional and structural characterization of the CmiS1 homolog SAV606 from MA-4680. analysis indicated that SAV606 catalyzes the Michael addition of glycine to crotonic acid thioester and subsequent hydrolysis yielding ()--carboxymethyl-3-aminobutyric acid. We also determined the crystal structures of SAV606 both in ligand-free form at 2.4 Å resolution and in complex with ()--carboxymethyl-3-aminobutyric acid at 2.0 Å resolution. We found that SAV606 adopts an α/β hotdog fold and has an active site at the dimeric interface. Examining the complexed structure, we noted that the substrate-binding loop comprising Tyr-53-Asn-61 recognizes the glycine moiety of ()--carboxymethyl-3-aminobutyric acid. Moreover, we found that SAV606 does not contain an acidic residue at the active site, which is distinct from canonical hotdog thioesterases. Site-directed mutagenesis experiments revealed that His-59 plays a crucial role in both the Michael addition and hydrolysis via a water molecule. These results allow us to propose the reaction mechanism of the SAV606-catalyzed Michael addition and thioester hydrolysis and provide new insight into the multiple functions of a thioesterase family enzyme.

Isonitrile Formation by a Non‐Heme Iron(II)‐Dependent Oxidase/Decarboxylase

“…However the recent biochemical and structural characterization of three ScoD homologues indicated that these thioesterases have dual functions with enzymatic hydrolysis occurring immediately after the Michael addition, both steps mediated by a single Gly residue in the active site. 10–12 These results raised the question of what the true substrate of ScoE is. We thus initiated an effort to reconstitute the in vitro activity of ScoE using various substrates that were chemically or chemoenzymatically synthesized.…”

Current Understanding toward Isonitrile Group Biosynthesis and Mechanism

“…[ 79‐80 ] Moreover, SAV606, a homolog to Rv0098, identified from S. avermitilis MA‐4680, has a dual function. [ 81 ] In addition to triggering the hydrolysis of the corresponding thioester to form N ‐carboxymethyl‐3‐aminononanoate ( 3 ), it also catalyzes the addition of glycine onto 24 (Figure 10B). Rv0099 , also known as FadD10, a ScoC homolog, likely serves a function of as fatty acyl‐AMP ligase.…”

Isonitrile Formation by a Non‐Heme Iron(II)‐Dependent Oxidase/Decarboxylase

“…The proposed pathway thus accounts for the necessity and sufficiency of these five core biosynthetic enzymes for INLP synthesis (Figure ). However the recent biochemical and structural characterization of three ScoD homologues indicated that these thioesterases have dual functions with enzymatic hydrolysis occurring immediately after the Michael addition, both steps mediated by a single Gly residue in the active site . These results raised the question of what the true substrate of ScoE is.…”