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 Streptomyces 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 macrolactam antibiotic 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 Streptomyces avermitilis MA-4680. In vitro analysis indicated that SAV606 catalyzes the Michael addition of glycine to crotonic acid thioester and subsequent hydrolysis yielding (R)-N-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 (R)-N-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 (R)-N-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.
Highlights
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
We found that SAV606 adopts an ␣/ hotdog fold and has an active site at the dimeric interface
We found that SAV606 does not contain an acidic residue at the active site, which is distinct from canonical hotdog thioesterases
Summary
Thioesterases (TEs) hydrolyze the thioester bond involved in acyl-acyl carrier protein (ACP) and acyl-CoA to release a carboxylic acid. The hotdog TEs, which belong to families 4 –15 and 24, are structurally and evolutionarily related to a family of dehydratases (DHs) [5, 7] They commonly hydrolyze the thioester bond of the acyl-CoA molecule, but some of them act on acyl-ACP. The family 24 TE Rv0098, which catalyzes the hydrolysis of a long-chain fatty acyl-CoA in Mycobacterium tuberculosis, is hypothesized to use two Tyr residues and a Asn residue for activation of the thioester carbonyl and stabilization. CmiS1, which belongs to the family 24 TEs, catalyzes the Michael addition of glycine to the -position of the non-2-enoyl-ACP and subsequent hydrolysis of the thioester to give N-carboxymethyl-3-aminononanoic acid (Fig. 1A). The structure of SAV606 in complex with the product molecule allows us to propose the mechanism of the Michael addition of glycine to an ␣,-unsaturated thioester
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