Determination of GDSL-subtrate complex in understanding the selectivity of novel GDSL esterase catalysis

GDSL esterase is designated as a member of Family II of lipolytic enzymes known to catalyse the synthesis and hydrolysis of ester bonds. The enzyme possesses a highly conserved motif Ser-Gly-Asn-His (SGNH) in the four conserved blocks I, II, III, and V respectively. Esterases perform synthesis by using a two-step mechanism where the nucleophilic Ser initiate the synthesis by first attacking the ester’s carbonyl carbon and produce an acyl enzyme intermediate and alcohol. Second, the catalytic His will facilitate the proton transfer from water which resulting in hydroxide attacks the carbonyl carbon and release of carboxylic acid. The enzyme’s characteristics such as region-, chemo- and enantioselectivity help in resolving the racemic mixture of singleisomer chiral drugs. To date, esterase enzyme identified from microbial sources have been widely used in various biotechnological applications such as manufacturing of biofuels and fine chemicals whereas identification of esterase enzyme in plant contributed to the regulation of plant hormone. The side effects of racemic mixture of commercially available naproxen can be overcome by resolving the racemic mixture of naproxen esters. In this study, determination of GDSL esterase and p-nitrophenyl butyrate which act as a substrate is identified to guide the protein engineering of GDSL esterase in resolving the racemic mixture of naproxen esters in the future. Recently, crystal structure of GDSL esterase from Photobacterium J15 has been reported (PDB ID: 5XTU) but not in complex with substrate. Therefore, GDSL in complex with substrate could provide insights into the binding mode of substrate toward inactive form of GDSL esterase (S12A) and identify the hot spot residues for the designing of a better binding pocket. Insight into molecular mechanisms is limited due to the lack of crystal structure of GDSL esterase-substrate complex. The crystallization of mutant GDSL esterase (S12A) and its complex with substrate are successfully reported. X-ray crystallography was performed in order to obtain the three-dimensional (3D) structure of the mutant GDSL esterase (S12A) and its complex with substrate. The recombinant Escherichia coli Rosetta-gami (DE3) pLysS harbouring mutant GDSL S12A was expressed at 20°C and induced by 0.1 mM IPTG. The expressed protein was then subjected to two-step of chromatography; affinity chromatography and ion-exchange chromatography. Prior to protein crystallisation, the affinity tags were removed of the purified protein and validated by SDS-PAGE analysis. The size of the mutant GDSL S12A is 36 kDa without affinity tags. The purified protein of the mutant GDSL S12A was crystallised in an optimized formulation containing 0.2 M ammonium acetate as salt, 30% (w/v) polyethylene glycol 4,000 as precipitant in 0.1 M sodium acetate trihydrate buffer at pH 4.6 with incubation at 15°C. For crystal of the mutant GDSL S12A, it was diffracted at 1.96 Å. As for mutant GDSL S12A substrate, the crystal was first soaked in pNP-butyrate solution prior to X-ray diffraction. The crystal of mutant GDSL S12A-substrate was diffracted at 1.73 Å. Both the crystals belong to orthorhombic space group of P212121. Analysis of the 3D structure of both structures showed that Ser12 of the catalytic triad has been mutated to Ala12. A DMSO molecule was found in the catalytic triad instead of the substrate pNP-butyrate. Further, a hydrolysed product of pNPbutyrate (i.e. butyric acid) was found far from the catalytic site of the mutant GDSL S12A. The substrate-bound structure has become vital in determining the hot spot residue for GDSL esterase. The solved structures aid in unveiling the interactions between the mutant GDSL S12A with substrate. The information could guide in the rational design of GDSL esterase in overcoming the medical limitations associated with racemic mixture.

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