5WSY

The complex structure of SAV606 with N-carboxymethyl-3-aminobutyrate

Summary for 5WSY

• Entry DOI: 10.2210/pdb5wsy/pdb
• Related: 5WSX
• Descriptor: Uncharacterized protein, (3~{R})-3-(2-hydroxy-2-oxoethylamino)butanoic acid (3 entities in total)
• Functional Keywords: thioesterase, hotdog fold, hydrolase
• Biological source: Streptomyces avermitilis (strain ATCC 31267 / DSM 46492 / JCM 5070 / NBRC 14893 / NCIMB 12804 / NRRL 8165 / MA-4680)
• Total number of polymer chains: 2
• Total formula weight: 39534.55

Authors

Chisuga, T.,Miyanaga, A.,Kudo, F.,Eguchi, T. (deposition date: 2016-12-08, release date: 2017-05-31, Last modification date: 2023-11-08)

Primary citation

Chisuga, T.,Miyanaga, A.,Kudo, F.,Eguchi, T.
Structural analysis of the dual-function thioesterase SAV606 unravels the mechanism of Michael addition of glycine to an alpha , beta-unsaturated thioester.
J. Biol. Chem., 292:10926-10937, 2017

PubMed 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 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 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.

PubMed: 28522606
DOI: 10.1074/jbc.M117.792549

Experimental method

X-RAY DIFFRACTION (2 Å)