Structural characterisation of Burkholderia pseudomallei BPSL0610 reveals its thioesterase features

Burkholderia pseudomallei (BP) is the aetiological agent of melioidosis with infections resulting in significant mortality in both animals and humans. BP is known to form biofilm which has been associated with relapse in melioidosis patients. Biofilms are an aggregation of microbial cells enclosed within a self-produced extracellular polymeric matrix. The bpsl0610 transcript was highly expressed during BP biofilm formation and the gene is present within a gene cluster (becA-R) that encodes the molecular machinery to produce exopolysaccharides. The BPSL0610 protein has thus been implicated in BP biofilm exopolysaccharide production. Since the specific function of BPSL0610 is unclear based on genome annotations, computational tools were utilised to determine the protein’s function. The three-dimensional structure of BPSL0610 was predicted using AlphaFold2 which revealed that the protein consists of two sequential canonical α/β hydrolase domains. Both domains resemble carboxylesterases (EC3.1.1.1) but only the C-terminal domain retained a conserved catalytic triad composed of Cys445, His604, and Asp571. This suggests that the cysteine may serve as a key catalytic residue in this enzyme, a feature commonly found in thioesterases. Esterase assays of the recombinant protein of BPSL0610 indeed displayed catalytic activity towards short chain ester substrates. Furthermore, the high confidence ternary complex structure of BPSL0610-BPSL0615 with the 4’-phosphopantetheine (4’-PP) arm on the acyl carrier protein (ACP) (BPSL0615) was predicted. The 4’-PP was shown to be positioned in the active site of BPSL0610 with similar orientation to other reported ternary complex structures of thioesterase and carrier protein with 4’-PP attached. This suggests that hydrolysis of the thioester bond of 4’-PP bound substrate may take place in the active site of BPSL0610. Taken together, the findings suggest that BPSL0610 is likely a thioesterase, which may function as part of the proposed molecular machinery that mediates exopolysaccharide synthesis and modification in BP.