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Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity

Ker DS, Chan KG, Othman R, Hassan M, Ng CL

Phytochemistry, 2020 May;173:112286.
PMID: 32059132 DOI: 10.1016/j.phytochem.2020.112286

The chemical formation of terpenes in nature is carried out by terpene synthases as the main biocatalysts to guide the carbocation intermediate to form structurally diverse compounds including acyclic, mono- and multiple cyclic products. Despite intensive study of the enzyme active site, the mechanism of specific terpene biosynthesis remains unclear. Here we demonstrate that a single mutation of the amino acid L454G or L454A in the active site of Persicaria minor β-sesquiphellandrene synthase leads to a more promiscuous enzyme that is capable of producing additional hydroxylated sesquiterpenes such as sesquicineole, sesquisabinene hydrate and α-bisabolol. Furthermore, the same L454 residue mutation (L454G or L454A) in the active site also improves the protein homogeneity compared to the wild type protein. Taken together, our results demonstrate that residue Leucine 454 in the active site of β-sesquiphellandrene synthase is important for sesquiterpene product diversity as well as the protein homogeneity in solution.
Fulltext Structural and functional analysis of Hydra Actinoporin-Like Toxin 1 (HALT-1)

Ker DS, Sha HX, Jonet MA, Hwang JS, Ng CL

Sci Rep, 2021 10 19;11(1):20649.
PMID: 34667248 DOI: 10.1038/s41598-021-99879-5

Actinoporins are a family of α-pore-forming toxins (α-PFTs) that have been identified in sea anemones. Recently, a freshwater Hydra Actinoporin-Like Toxin (HALT) gene family was found in Hydra magnipapillata. Unlike sea anemone actinoporins that use sphingomyelin as their main recognition target, the HALTs proteins may recognise alternative lipid molecules as their target. To unveil the structural insights into lipid preference of HALTs protein as compared to sea anemone actinoporins, we have determined the first crystal structure of actinoporin-like toxin, HALT-1 at 1.43 Å resolution with an acetylated lysine residue K76. Despite the overall structure of HALT-1 sharing a high structural similarity to sea anemone actinoporins, the atomic resolution structure revealed several unique structural features of HALT-1 that may influence the lipid preference and oligomerisation interface. The HALT-1 contains a RAG motif in place of the highly conserved RGD motif found in sea anemone actinoporins. The RAG motif contributed to a sharper β9-β10 turn, which may sway its oligomerisation interface in comparison to sea anemone actinoporins. In the lipid-binding region, the HALT-1 contains a shorter α2 helix and a longer α2-β9 loop due to deletion and subsequently an insertion of five amino acid residues in comparison to the sea anemone actinoporins. Structure comparison and molecular docking analysis further revealed that the HALT-1 lipid-binding site may favour sphingolipids with sulfate or phosphate head group more than the sphingomyelin. The structure of HALT-1 reported here provides a new insight for a better understanding of the evolution and lipid recognition mechanism of actinoporin.
Fulltext Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase

Ker DS, Pang SL, Othman NF, Kumaran S, Tan EF, Krishnan T, et al.

PeerJ, 2017;5:e2961.
PMID: 28265494 DOI: 10.7717/peerj.2961

BACKGROUND: Sesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment.
METHODS: The enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server.
RESULTS: Our findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases.
DISCUSSION: The study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.
Fulltext Structural and functional analyses of Burkholderia pseudomallei BPSL1038 reveal a Cas-2/VapD nuclease sub-family

Shaibullah S, Shuhaimi N, Ker DS, Mohd-Sharif N, Ho KL, Teh AH, et al.

Commun Biol, 2023 Sep 08;6(1):920.
PMID: 37684342 DOI: 10.1038/s42003-023-05265-4

Burkholderia pseudomallei is a highly versatile pathogen with ~25% of its genome annotated to encode hypothetical proteins. One such hypothetical protein, BPSL1038, is conserved across seven bacterial genera and 654 Burkholderia spp. Here, we present a 1.55 Å resolution crystal structure of BPSL1038. The overall structure folded into a modified βαββαβα ferredoxin fold similar to known Cas2 nucleases. The Cas2 equivalent catalytic aspartate (D11) pairs are conserved in BPSL1038 although B. pseudomallei has no known CRISPR associated system. Functional analysis revealed that BPSL1038 is a nuclease with endonuclease activity towards double-stranded DNA. The DNase activity is divalent ion independent and optimum at pH 6. The concentration of monovalent ions (Na+ and K+) is crucial for nuclease activity. An active site with a unique D11(X20)SST motif was identified and proposed for BPSL1038 and its orthologs. Structure modelling indicates the catalytic role of the D11(X20)SST motif and that the arginine residues R10 and R30 may interact with the nucleic acid backbone. The structural similarity of BPSL1038 to Cas2 proteins suggests that BPSL1038 may represent a sub-family of nucleases that share a common ancestor with Cas2.