Publications by authors named "Chyan Leong Ng"

25 Publications

  • Page 1 of 1

Purification, biochemical characterisation and bioinformatic analysis of recombinant farnesol dehydrogenase from Theobroma cacao.

Plant Physiol Biochem 2021 Apr 5;161:143-155. Epub 2021 Feb 5.

Institute of Systems Biology, Universiti Kebangsaan Malaysia, UKM Bangi, Bangi, Selangor, 43600, Malaysia. Electronic address:

The juvenile hormones (JH) in plants are suggested to act as a form of plant defensive strategy especially against insect herbivory. The oxidation of farnesol to farnesoic acid is a key step in the juvenile hormone biosynthesis pathway. We herein present the purification and characterisation of the recombinant Theobroma cacao farnesol dehydrogenase enzyme that catalyses oxidation of farnesol to farnesal. The recombinant enzyme was purified to apparent homogeneity by affinity chromatography. The purified enzyme was characterised in terms of its deduced amino acid sequences, phylogeny, substrate specificity, kinetic parameters, structural modeling, and docking simulation. The phylogenetic analysis indicated that the T. cacao farnesol dehydrogenase (TcFolDH) showed a close relationship with A. thaliana farnesol dehydrogenase gene. The TcFolDH monomer had a large N-terminal domain which adopted a typical Rossmann-fold, harboring the GxxGxG motif (NADP(H)-binding domain) and a small C-terminal domain. The enzyme was a homotrimer comprised of subunits with molecular masses of 36 kDa. The TcFolDH was highly specific to NADP as coenzyme. The substrate specificity studies showed trans, trans-farnesol was the most preferred substrate for the TcFolDH, suggesting that the purified enzyme was a NADP-dependent farnesol dehydrogenase. The docking of trans, trans-farnesol and NADP into the active site of the enzyme showed the important residues, and their interactions involved in the substrate and coenzyme binding of TcFolDH. Considering the extensive involvement of JH in both insects and plants, an in-depth knowledge on the recombinant production of intermediate enzymes of the JH biosynthesis pathway could help provide a potential method for insect control.
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http://dx.doi.org/10.1016/j.plaphy.2021.01.050DOI Listing
April 2021

IgE-binding residues analysis of the house dust mite allergen Der p 23.

Sci Rep 2021 Jan 13;11(1):921. Epub 2021 Jan 13.

Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore.

House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P < 0.001). Atopic individuals who were only sensitized to HDM have a significantly higher number of IgE-binding residues than the individuals who were polysensitized to HDM and other crude allergens (P < 0.05). Individuals with allergic multimorbidity and moderate-to-severe allergic rhinitis also have a higher number of IgE-binding residues compared to those with single allergic disease and mild allergic rhinitis. The results prompt us to hypothesize that the individuals who have a higher number of IgE-binding residues may face a bigger challenge to be treated through immunotherapy due to the complexity in designing an effective hypoallergen with a high number of IgE-binding residues. We propose that the development of a refined molecular diagnostic assay, which includes alanine substitution of surface-exposed residues could be a more precise diagnostic strategy to identify all the IgE-binding residues of a major allergen for an atopic individual and the development could be another new dimension in allergy diagnosis and allergen immunotherapy treatment.
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http://dx.doi.org/10.1038/s41598-020-79820-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806929PMC
January 2021

Aspergillus fumigatus AR04 obeys Arrhenius' rule in cultivation temperature shifts from 30 to 40°C.

Microb Biotechnol 2021 Jan 9. Epub 2021 Jan 9.

Institute of Biotechnology, Brandenburg University of Technology Cottbus - Senftenberg, Universitätsplatz 1, Senftenberg, D-01958, Germany.

To set a benchmark in fungal growth rate, a differential analysis of prototrophic Aspergillus fumigatus AR04 with three ascomycetes applied in > 10 t year scale was performed, i.e. Ashbya gosspyii (riboflavin), Aspergillus niger (citric acid) and Aspergillus oryzae (food-processing). While radial colony growth decreased 0.5-fold when A. gossypii was cultivated at 40°C instead of 28°C, A. fumigatus AR04 responded with 1.7-fold faster hyphal growth. A. niger and A. oryzae formed colonies at 40°C, but not at 43°C. Moreover, all A. fumigatus strains tested grew even at 49°C. In chemostat experiments, A. fumigatus AR04 reached steady state at a dilution rate of 0.7 h at 40°C, 120% more than reported for A. gossypii at 28°C. To study mycelial growth rates under unlimited conditions, carbon dioxide increase rates were calculated from concentrations detected online in the exhaust of batch fermentations for 3 h only. All rates calculated suggest that A. fumigatus AR04 approximates Arrhenius' rule when comparing short cultivations at 30°C with those at 40°C. Linearization of the exponential phase and comparison of the slopes revealed an increase to 192% by the 10°C up-shift.
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http://dx.doi.org/10.1111/1751-7915.13739DOI Listing
January 2021

Comparative metabolomics of Phialemonium curvatum as an omnipotent fungus cultivated on crude palm oil versus glucose.

Microb Cell Fact 2020 Sep 9;19(1):179. Epub 2020 Sep 9.

Institute of Systems Biology, Universiti Kebangsaan Malaysia (UKM), 43600, Bangi, Selangor, Malaysia.

Background: Sugars and triglycerides are common carbon sources for microorganisms. Nonetheless, a systematic comparative interpretation of metabolic changes upon vegetable oil or glucose as sole carbon source is still lacking. Selected fungi that can grow in acidic mineral salt media (MSM) with vegetable oil had been identified recently. Hence, this study aimed to investigate the overall metabolite changes of an omnipotent fungus and to reveal changes at central carbon metabolism corresponding to both carbon sources.

Results: Targeted and non-targeted metabolomics for both polar and semi-polar metabolites of Phialemonium curvatum AWO2 (DSM 23903) cultivated in MSM with palm oil (MSM-P) or glucose (MSM-G) as carbon sources were obtained. Targeted metabolomics on central carbon metabolism of tricarboxylic acid (TCA) cycle and glyoxylate cycle were analysed using LC-MS/MS-TripleQ and GC-MS, while untargeted metabolite profiling was performed using LC-MS/MS-QTOF followed by multivariate analysis. Targeted metabolomics analysis showed that glyoxylate pathway and TCA cycle were recruited at central carbon metabolism for triglyceride and glucose catabolism, respectively. Significant differences in organic acids concentration of about 4- to 8-fold were observed for citric acid, succinic acid, malic acid, and oxaloacetic acid. Correlation of organic acids concentration and key enzymes involved in the central carbon metabolism was further determined by enzymatic assays. On the other hand, the untargeted profiling revealed seven metabolites undergoing significant changes between MSM-P and MSM-G cultures.

Conclusions: Overall, this study has provided insights on the understanding on the effect of triglycerides and sugar as carbon source in fungi global metabolic pathway, which might become important for future optimization of carbon flux engineering in fungi to improve organic acids production when vegetable oil is applied as the sole carbon source.
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http://dx.doi.org/10.1186/s12934-020-01434-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487481PMC
September 2020

In silico analysis on the functional and structural impact of Rad50 mutations involved in DNA strand break repair.

PeerJ 2020 22;8:e9197. Epub 2020 May 22.

Department of Food Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.

Background: DNA double strand break repair is important to preserve the fidelity of our genetic makeup after DNA damage. Rad50 is one of the components in MRN complex important for DNA repair mechanism. Rad50 mutations can lead to microcephaly, mental retardation and growth retardation in human. However, Rad50 mutations in human and other organisms have never been gathered and heuristically compared for their deleterious effects. It is important to assess the conserved region in Rad50 and its homolog to identify vital mutations that can affect functions of the protein.

Method: In this study, Rad50 mutations were retrieved from SNPeffect 4.0 database and literature. Each of the mutations was analyzed using various bioinformatic analyses such as PredictSNP, MutPred, SNPeffect 4.0, I-Mutant and MuPro to identify its impact on molecular mechanism, biological function and protein stability, respectively.

Results: We identified 103 mostly occurred mutations in the Rad50 protein domains and motifs, which only 42 mutations were classified as most deleterious. These mutations are mainly situated at the specific motifs such as Walker A, Q-loop, Walker B, D-loop and signature motif of the Rad50 protein. Some of these mutations were predicted to negatively affect several important functional sites that play important roles in DNA repair mechanism and cell cycle signaling pathway, highlighting Rad50 crucial role in this process. Interestingly, mutations located at non-conserved regions were predicted to have neutral/non-damaging effects, in contrast with previous experimental studies that showed deleterious effects. This suggests that software used in this study may have limitations in predicting mutations in non-conserved regions, implying further improvement in their algorithm is needed. In conclusion, this study reveals the priority of acid substitution associated with the genetic disorders. This finding highlights the vital roles of certain residues such as K42E, C681A/S, CC684R/S, S1202R, E1232Q and D1238N/A located in Rad50 conserved regions, which can be considered for a more targeted future studies.
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http://dx.doi.org/10.7717/peerj.9197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247530PMC
May 2020

Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity.

Phytochemistry 2020 May 12;173:112286. Epub 2020 Feb 12.

Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, UKM, Bangi, Selangor, 43600, Malaysia. Electronic address:

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.
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http://dx.doi.org/10.1016/j.phytochem.2020.112286DOI Listing
May 2020

Crystal structure and epitope analysis of house dust mite allergen Der f 21.

Sci Rep 2019 03 20;9(1):4933. Epub 2019 Mar 20.

Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.

Group 21 and 5 allergens are homologous house dust mite proteins known as mid-tier allergens. To reveal the biological function of group 21 allergens and to understand better the allergenicity of the rDer f 21 allergen, we determined the 1.5 Å crystal structure of rDer f 21 allergen from Dermatophagoides farinae. The rDer f 21 protein consists of a three helical bundle, similar to available structures of group 21 and homologous group 5 allergens. The rDer f 21 dimer forms a hydrophobic binding pocket similar to the one in the Der p 5 allergen, which indicates that both of the homologous groups could share a similar function. By performing structure-guided mutagenesis, we mutated all 38 surface-exposed polar residues of the rDer f 21 allergen and carried out immuno-dot blot assays using 24 atopic sera. Six residues, K10, K26, K42, E43, K46, and K48, which are located in the region between the N-terminus and the loop 1 of rDer f 21 were identified as the major IgE epitopes of rDer f 21. Epitope mapping of all potential IgE epitopes on the surface of the rDer f 21 crystal structure revealed heterogeneity in the sIgE recognition of the allergen epitopes in atopic individuals. The higher the allergen-sIgE level of an individual, the higher the number of epitope residues that are found in the allergen. The results illustrate the clear correlation between the number of specific major epitope residues in an allergen and the sIgE level of the atopic population.
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http://dx.doi.org/10.1038/s41598-019-40879-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426935PMC
March 2019

Functional Genomics.

Adv Exp Med Biol 2018;1102:11-30

Institute of Systems Biology, Universiti Kebangsaan Malaysia (UKM), Bangi, Malaysia.

Functional genomics encompasses diverse disciplines in molecular biology and bioinformatics to comprehend the blueprint, regulation, and expression of genetic elements that define the physiology of an organism. The deluge of sequencing data in the postgenomics era has demanded the involvement of computer scientists and mathematicians to create algorithms, analytical software, and databases for the storage, curation, and analysis of biological big data. In this chapter, we discuss on the concept of functional genomics in the context of systems biology and provide examples of its application in human genetic disease studies, molecular crop improvement, and metagenomics for antibiotic discovery. An overview of transcriptomics workflow and experimental considerations is also introduced. Lastly, we present an in-house case study of transcriptomics analysis of an aromatic herbal plant to understand the effect of elicitation on the biosynthesis of volatile organic compounds.
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http://dx.doi.org/10.1007/978-3-319-98758-3_2DOI Listing
July 2019

Crystal structure and functional analysis of human C1ORF123.

PeerJ 2018 28;6:e5377. Epub 2018 Sep 28.

Institute of Systems Biology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.

Proteins of the DUF866 superfamily are exclusively found in eukaryotic cells. A member of the DUF866 superfamily, C1ORF123, is a human protein found in the open reading frame 123 of chromosome 1. The physiological role of C1ORF123 is yet to be determined. The only available protein structure of the DUF866 family shares just 26% sequence similarity and does not contain a zinc binding motif. Here, we present the crystal structure of the recombinant human C1ORF123 protein (rC1ORF123). The structure has a 2-fold internal symmetry dividing the monomeric protein into two mirrored halves that comprise of distinct electrostatic potential. The N-terminal half of rC1ORF123 includes a zinc-binding domain interacting with a zinc ion near to a potential ligand binding cavity. Functional studies of human C1ORF123 and its homologue in the fission yeast (SpEss1) point to a role of DUF866 protein in mitochondrial oxidative phosphorylation.
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http://dx.doi.org/10.7717/peerj.5377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166629PMC
September 2018

Metabolomics data of leaf using LC-ESI-TOF-MS.

Data Brief 2018 Jun 4;18:1212-1216. Epub 2018 Apr 4.

Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia (UKM), 43600 Bangi, Selangor, Malaysia.

is a psychoactive plant known as "ketum" in Malaysia and "kratom" in Thailand. This plant is distinctly known to produce two important alkaloids, namely mitragynine (MG) and 7-hydroxymitragynine (7-OH-MG) that can bind to opioid receptors [1]. MG was reported to exhibit antidepressant properties in animal studies [2]. These compounds were also proposed to have the potential to replace opioid analgesics with much lower risks of side effects [3]. To date, there are only over 40 metabolites identified in [4,5]. To obtain a more complete profile of secondary metabolites in ketum, we performed metabolomics study using mature leaves of the green variety. The leaf samples were extracted using methanol prior to liquid chromatography-electrospray ionization-time of flight-mass spectrometry (LC-ESI-TOF-MS) analysis. This data can be useful to for the identification of unknown metabolites that are associated with alkaloid biosynthesis pathway in .
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http://dx.doi.org/10.1016/j.dib.2018.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996727PMC
June 2018

Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

Plant Physiol Biochem 2018 Feb 26;123:359-368. Epub 2017 Dec 26.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia; Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia. Electronic address:

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp, Leu and Ala). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.
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http://dx.doi.org/10.1016/j.plaphy.2017.12.033DOI Listing
February 2018

Genomic characterization of a new endophytic identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production.

PeerJ 2017 29;5:e3738. Epub 2017 Nov 29.

School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.

Background: are well known for their capability to produce many bioactive secondary metabolites with medical and industrial importance. Here we report a novel bioactive phenazine compound, 6-((2-hydroxy-4-methoxyphenoxy) carbonyl) phenazine-1-carboxylic acid (HCPCA) extracted from , an endophyte isolated from the ethnomedicinal

Methods: The HCPCA chemical structure was determined using nuclear magnetic resonance spectroscopy. We conducted whole genome sequencing for the identification of the gene cluster(s) believed to be responsible for phenazine biosynthesis in order to map its corresponding pathway, in addition to bioinformatics analysis to assess the potential of in producing other useful secondary metabolites.

Results: The genome comprises an 8,328,719 bp linear chromosome with high GC content (71.35%) consisting of 12 rRNA operons, 81 tRNA, and 7,558 protein coding genes. We identified 24 gene clusters involved in polyketide, nonribosomal peptide, terpene, bacteriocin, and siderophore biosynthesis, as well as a gene cluster predicted to be responsible for phenazine biosynthesis.

Discussion: The HCPCA phenazine structure was hypothesized to derive from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid and 4-methoxybenzene-1,2-diol, originated from the shikimic acid pathway. The identification of a biosynthesis pathway gene cluster for phenazine antibiotics might facilitate future genetic engineering design of new synthetic phenazine antibiotics. Additionally, these findings confirm the potential of for producing various antibiotics and secondary metabolites.
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http://dx.doi.org/10.7717/peerj.3738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712208PMC
November 2017

Whole-genome shotgun sequence of phenazine-producing endophytic SUK12.

Genom Data 2017 Sep 24;13:7-10. Epub 2017 May 24.

School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.

sp. produces bioactive compounds with a broad spectrum of activities. SUK12 has been identified as a novel endophytic bacteria isolated from ethnomedicinal plant , and was found to produce the phenazine class of biologically active antimicrobial metabolites. The potential use of the phenazines has led to our research interest in determining the genome sequence of SUK12. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number PRJNA269542. The raw sequence data are available [https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP105770].
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http://dx.doi.org/10.1016/j.gdata.2017.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5447654PMC
September 2017

Purification and biochemical characterization of recombinant  -sesquiphellandrene synthase.

PeerJ 2017 28;5:e2961. Epub 2017 Feb 28.

Institute of Systems Biology, Universiti Kebangsaan Malaysia , Bangi , Selangor , Malaysia.

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 . Here, we report the expression, purification and characterization of recombinant 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 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 C 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 -sesquiphellandrene as a major product and -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, and 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.
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http://dx.doi.org/10.7717/peerj.2961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333544PMC
February 2017

Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative γ-butyrolactone receptor protein from Streptomyces fradiae.

Acta Crystallogr F Struct Biol Commun 2017 02 31;73(Pt 2):109-115. Epub 2017 Jan 31.

Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.

TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P321, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
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http://dx.doi.org/10.1107/S2053230X17001212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297932PMC
February 2017

Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant.

PLoS One 2016 25;11(8):e0161707. Epub 2016 Aug 25.

Institute of Systems Biology, Universiti Kebangsaan Malaysia (UKM), 43600 UKM, Bangi, Selangor, Malaysia.

Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards farnesal. Thus, it was suggested that this novel enzyme may be functioning specifically to oxidize farnesal in the later steps of JH III pathway. This report provides a basic understanding for recombinant production of this particular enzyme. Other strategies such as adding His-tag to the protein makes easy the purification of the protein which is completely different to the native protein. Complete sequence, structure and functional analysis of the enzyme will be important for developing insect-resistant crop plants by deployment of transgenic plant.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161707PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999093PMC
July 2017

A/T Run Geometry of B-form DNA Is Independent of Bound Methyl-CpG Binding Domain, Cytosine Methylation and Flanking Sequence.

Sci Rep 2016 08 9;6:31210. Epub 2016 Aug 9.

Department of Pathology, Faculty of Medicine and Health Sciences; Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.

DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed.
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http://dx.doi.org/10.1038/srep31210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4977486PMC
August 2016

Characterization of a type I pullulanase from Anoxybacillus sp. SK3-4 reveals an unusual substrate hydrolysis.

Appl Microbiol Biotechnol 2016 Jul 22;100(14):6291-6307. Epub 2016 Mar 22.

Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.

Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0-6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60 °C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26-72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362-370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme.
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http://dx.doi.org/10.1007/s00253-016-7451-6DOI Listing
July 2016

Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass.

Sci Rep 2016 Mar 15;6:23126. Epub 2016 Mar 15.

Universiti Kebangsaan Malaysia, Institute of Systems Biology, 43600 UKM Bangi, Selangor, Malaysia.

A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.
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http://dx.doi.org/10.1038/srep23126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791539PMC
March 2016

Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein.

Acta Crystallogr F Struct Biol Commun 2016 Mar 16;72(Pt 3):207-13. Epub 2016 Feb 16.

Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.

C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.
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http://dx.doi.org/10.1107/S2053230X16002016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774879PMC
March 2016

Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) from Dermatophagoides farinae.

Acta Crystallogr F Struct Biol Commun 2015 Nov 23;71(Pt 11):1396-400. Epub 2015 Oct 23.

Institute of Systems Biology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia.

Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
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http://dx.doi.org/10.1107/S2053230X1501818XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631589PMC
November 2015

Crystallization and preliminary crystallographic studies of the hypothetical protein BPSL1038 from Burkholderia pseudomallei.

Acta Crystallogr F Struct Biol Commun 2014 Dec 28;70(Pt 12):1697-700. Epub 2014 Nov 28.

Institute of Systems Biology, Universiti Kebangsaan Malaysia (UKM), Bangi, Selangor 43600, Malaysia.

Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ∼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55 Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73 Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%.
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http://dx.doi.org/10.1107/S2053230X14025278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4259243PMC
December 2014

Unusual base pairing during the decoding of a stop codon by the ribosome.

Nature 2013 Aug 30;500(7460):107-10. Epub 2013 Jun 30.

MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

During normal translation, the binding of a release factor to one of the three stop codons (UGA, UAA or UAG) results in the termination of protein synthesis. However, modification of the initial uridine to a pseudouridine (Ψ) allows efficient recognition and read-through of these stop codons by a transfer RNA (tRNA), although it requires the formation of two normally forbidden purine-purine base pairs. Here we determined the crystal structure at 3.1 Å resolution of the 30S ribosomal subunit in complex with the anticodon stem loop of tRNA(Ser) bound to the ΨAG stop codon in the A site. The ΨA base pair at the first position is accompanied by the formation of purine-purine base pairs at the second and third positions of the codon, which show an unusual Watson-Crick/Hoogsteen geometry. The structure shows a previously unsuspected ability of the ribosomal decoding centre to accommodate non-canonical base pairs.
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http://dx.doi.org/10.1038/nature12302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732562PMC
August 2013

Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin.

Proc Natl Acad Sci U S A 2012 Jul 14;109(27):10984-9. Epub 2012 Jun 14.

Institut für Medizinische Mikrobiologie, Universität Zürich, 8006 Zürich, Switzerland.

Aminoglycosides are potent antibacterials, but therapy is compromised by substantial toxicity causing, in particular, irreversible hearing loss. Aminoglycoside ototoxicity occurs both in a sporadic dose-dependent and in a genetically predisposed fashion. We recently have developed a mechanistic concept that postulates a key role for the mitochondrial ribosome (mitoribosome) in aminoglycoside ototoxicity. We now report on the surprising finding that apramycin, a structurally unique aminoglycoside licensed for veterinary use, shows little activity toward eukaryotic ribosomes, including hybrid ribosomes which were genetically engineered to carry the mitoribosomal aminoglycoside-susceptibility A1555G allele. In ex vivo cultures of cochlear explants and in the in vivo guinea pig model of chronic ototoxicity, apramycin causes only little hair cell damage and hearing loss but it is a potent antibacterial with good activity against a range of clinical pathogens, including multidrug-resistant Mycobacterium tuberculosis. These data provide proof of concept that antibacterial activity can be dissected from aminoglycoside ototoxicity. Together with 3D structures of apramycin-ribosome complexes at 3.5-Å resolution, our results provide a conceptual framework for further development of less toxic aminoglycosides by hypothesis-driven chemical synthesis.
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http://dx.doi.org/10.1073/pnas.1204073109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390888PMC
July 2012

Crystal structure of Mil (Mth680): internal duplication and similarity between the Imp4/Brix domain and the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases.

EMBO Rep 2005 Feb;6(2):140-6

York Structural Biology Laboratory, Chemistry Department, University of York, York YO10 5YW, UK.

Proteins of the Imp4/Brix superfamily are involved in ribosomal RNA processing, an essential function in all cells. We report the first structure of an Imp4/Brix superfamily protein, the Mil (for Methanothermobacter thermautotrophicus Imp4-like) protein (gene product Mth680), from the archaeon M. thermautotrophicus. The amino- and carboxy-terminal halves of Mil show significant structural similarity to one another, suggesting an origin by means of an ancestral duplication. Both halves show the same fold as the anticodon-binding domain of class IIa aminoacyl-tRNA synthetases, with greater conservation seen in the N-terminal half. This structural similarity, together with the charge distribution in Mil, suggests that Imp4/Brix superfamily proteins could bind single-stranded segments of RNA along a concave surface formed by the N-terminal half of their beta-sheet and a central alpha-helix. The crystal structure of Mil is incompatible with the presence, in the Imp4/Brix domain, of a helix-turn-helix motif that was proposed to comprise the RNA-binding moiety of the Imp4/Brix proteins.
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http://dx.doi.org/10.1038/sj.embor.7400328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299238PMC
February 2005