Displaying publications 1 - 20 of 38 in total

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  1. Halophiles: biology, adaptation, and their role in decontamination of hypersaline environments
    Edbeib MF, Wahab RA, Huyop F
    World J Microbiol Biotechnol, 2016 Aug;32(8):135.
    PMID: 27344438 DOI: 10.1007/s11274-016-2081-9
    The unique cellular enzymatic machinery of halophilic microbes allows them to thrive in extreme saline environments. That these microorganisms can prosper in hypersaline environments has been correlated with the elevated acidic amino acid content in their proteins, which increase the negative protein surface potential. Because these microorganisms effectively use hydrocarbons as their sole carbon and energy sources, they may prove to be valuable bioremediation agents for the treatment of saline effluents and hypersaline waters contaminated with toxic compounds that are resistant to degradation. This review highlights the various strategies adopted by halophiles to compensate for their saline surroundings and includes descriptions of recent studies that have used these microorganisms for bioremediation of environments contaminated by petroleum hydrocarbons. The known halotolerant dehalogenase-producing microbes, their dehalogenation mechanisms, and how their proteins are stabilized is also reviewed. In view of their robustness in saline environments, efforts to document their full potential regarding remediation of contaminated hypersaline ecosystems merits further exploration.
  2. Fulltext l-2-Haloacid dehalogenase (DehL) from Rhizobium sp. RC1
    Adamu A, Wahab RA, Huyop F
    Springerplus, 2016;5(1):695.
    PMID: 27347470 DOI: 10.1186/s40064-016-2328-9
    l-2-Haloacid dehalogenase (DehL) from Rhizobium sp. RC1 is a stereospecific enzyme that acts exclusively on l-isomers of 2-chloropropionate and dichloroacetate. The amino acid sequence of this enzyme is substantially different from those of other l-specific dehalogenases produced by other organisms. DehL has not been crystallised, and hence its three-dimensional structure is unavailable. Herein, we review what is known concerning DehL and tentatively identify the amino acid residues important for catalysis based on a comparative structural and sequence analysis with well-characterised l-specific dehalogenases.
  3. Synthesis of geranyl propionate in a solvent-free medium using Rhizomucor miehei lipase covalently immobilized on chitosan-graphene oxide beads
    Isah AA, Mahat NA, Jamalis J, Attan N, Zakaria II, Huyop F, et al.
    Prep Biochem Biotechnol, 2017 Feb 07;47(2):199-210.
    PMID: 27341522 DOI: 10.1080/10826068.2016.1201681
    The chemical route of producing geranyl propionate involves the use of toxic chemicals, liberation of unwanted by-products as well as problematic separation process. In view of such problems, the use of Rhizomucor miehei lipase (RML) covalently bound onto activated chitosan-graphene oxide (RML-CS/GO) support is suggested. Following analyses using Fourier transform infrared spectroscopy, field emission scanning electron microscopy, transmission electron microscopy, and thermogravimetry, properties of the RML-CS/GO were characterized. A response surface methodological approach using a 3-level-four-factor (incubation time, temperature, substrate molar ratio, and stirring rate) Box-Behnken design was used to optimize the experimental conditions to maximize the yield of geranyl propionate. Results revealed that 76 ± 0.02% of recovered protein had yielded 7.2 ± 0.04 mg g(-1) and 211 ± 0.3% U g(-1) of the maximum protein loading and esterification activity, respectively. The actual yield of geranyl propionate (49.46%) closely agreed with the predicted value (49.97%) under optimum reaction conditions (temperature: 37.67°C, incubation time: 10.20 hr, molar ratio (propionic acid:geraniol): 1:3.28, and stirring rate: 100.70 rpm) and hence, verifying the suitability of this approach. Since the method is performed under mild conditions, the RML-CS/GO biocatalyst may prove to be an environmentally benign alternative for producing satisfactory yield of geranyl propionate.
  4. Fulltext An S188V mutation alters substrate specificity of non-stereospecific α-haloalkanoic acid dehalogenase E (DehE)
    Hamid AA, Hamid TH, Wahab RA, Omar MS, Huyop F
    PLoS One, 2015;10(3):e0121687.
    PMID: 25816329 DOI: 10.1371/journal.pone.0121687
    The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3-chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by Km. This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.
  5. Fulltext Physicochemical and antioxidant properties of Apis cerana honey from Lombok and Bali Islands
    Huyop F, Ullah S, Abdul Wahab R, Huda N, Sujana IGA, Saloko S, et al.
    PLoS One, 2024;19(4):e0301213.
    PMID: 38578814 DOI: 10.1371/journal.pone.0301213
    Limited honey production worldwide leads to higher market prices, thus making it prone to adulteration. Therefore, regular physicochemical analysis is imperative for ensuring authenticity and safety. This study describes the physicochemical and antioxidant properties of Apis cerana honey sourced from the islands of Lombok and Bali, showing their unique regional traits. A comparative analysis was conducted on honey samples from Lombok and Bali as well as honey variety from Malaysia. Moisture content was found slightly above 20% in raw honey samples from Lombok and Bali, adhering to the national standard (SNI 8664:2018) of not exceeding 22%. Both honey types displayed pH values within the acceptable range (3.40-6.10), ensuring favorable conditions for long-term storage. However, Lombok honey exhibited higher free acidity (78.5±2.14 meq/kg) than Bali honey (76.0±1.14 meq/kg), surpassing Codex Alimentarius recommendations (≤50 meq/kg). The ash content, reflective of inorganic mineral composition, was notably lower in Lombok (0.21±0.02 g/100) and Bali honey (0.14±0.01 g/100) compared to Tualang honey (1.3±0.02 g/100). Electric conductivity, indicative of mineral content, revealed Lombok and Bali honey with lower but comparable values than Tualang honey. Hydroxymethylfurfural (HMF) concentrations in Lombok (14.4±0.11 mg/kg) and Bali (17.6±0.25 mg/kg) were slightly elevated compared to Tualang honey (6.4±0.11 mg/kg), suggesting potential processing-related changes. Sugar analysis revealed Lombok honey with the highest sucrose content (2.39±0.01g/100g) and Bali honey with the highest total sugar content (75.21±0.11 g/100g). Both honeys exhibited lower glucose than fructose content, aligning with Codex Alimentarius guidelines. The phenolic content, flavonoids, and antioxidant activity were significantly higher in Lombok and Bali honey compared to Tualang honey, suggesting potential health benefits. Further analysis by LC-MS/MS-QTOF targeted analysis identified various flavonoids/flavanols and polyphenolic/phenolic acid compounds in Lombok and Bali honey. The study marks the importance of characterizing the unique composition of honey from different regions, ensuring quality and authenticity in the honey industry.
  6. Fulltext Exploring the genome of Lactobacillaceae spp. Sy-1 isolated from Heterotrigona itama honey
    Syed Yaacob SN, Huyop F, Misson M, Abdul Wahab R, Huda N
    PeerJ, 2022;10:e13053.
    PMID: 35345581 DOI: 10.7717/peerj.13053
    BACKGROUND: Honey produced by Heterotrigona itama is highly preferred among consumers due to its high-value as a functional food and beneficial lactic acid bacteria (LAB) reservoir. Fructophilic lactic acid bacteria (FLAB) are a group of LAB with unique growth characteristics and are regarded as promising producers of bioactive compounds. Hence, it is not surprising that LAB, especially FLAB, may be involved with the excellent bioactivity of H. itama honey. With the trending consumer preference for H. itama honey coupled with increasing awareness for healthy food, the genomic background of FLAB isolated from this honey must, therefore, be clearly understood. In this study, one FLAB strain designated as Sy-1 was isolated from freshly collected H. itama honey. Its FLAB behavior and genomic features were investigated to uncover functional genes that could add value to functional food.

    METHODS: The fructophilic characteristics of strain Sy-1 were determined, and the genome was sequenced using Illumina iSeq100 and Oxford Nanopore. The average nucleotide identity and phylogenetic analyses based on 16S rRNA, 92 core genes, and whole-genome sequence were performed to unravel the phylogenetic position of strain Sy-1. NCBI Prokaryotic Genome Annotation Pipeline annotated the genome, while the EggNOG-mapper, BLASTKoala, and GHOSTKoala were used to add functional genes and pathways information.

    RESULTS: Strain Sy-1 prefers D-fructose over D-glucose and actively metabolizes D-glucose in the presence of electron acceptors. Genomic annotation of strain Sy-1 revealed few genes involved in carbohydrate transport and metabolism, and partial deletion of adhE gene, in line with the characteristic of FLAB. The 16S rRNA gene sequence of strain Sy-1 showed the highest similarity to unknown LAB species isolated from the gut of honeybees. The phylogenetic analyses discovered that strain Sy-1 belonged to the Lactobacillaceae family and formed a separate branch closer to type strain from the genera of Acetilactobacillus and Apilactobacillus. The ANI analysis showed the similarity of the closest relative, Apilactobacillus micheneri Hlig3T. The assembled genome of Sy-1 contains 3 contigs with 2.03 Mbp and a 41% GC content. A total of 1,785 genes were identified, including 1,685 protein-coding genes, 68 tRNA, and 15 rRNA. Interestingly, strain Sy-1 encoded complete genes for the biosynthesis of folate and riboflavin. High-performance liquid chromatography analysis further confirmed the high production of folic acid (1.346 mg/L) by Sy-1.

    DISCUSSION: Based on phylogenetic and biochemical characteristics, strain Sy-1 should be classified as a novel genus in the family of Lactobacillaceae and a new member of FLAB. The genome information coupled with experimental studies supported the ability of strain Sy-1 to produce high folic acid. Our collective findings support the suitable application of FLAB strain Sy-1 in the functional food and pharmaceutical industries.

  7. Fulltext Customised structural, optical and antibacterial characteristics of cinnamon nanoclusters produced inside organic solvent using 532 nm Q-switched Nd:YAG-pulse laser ablation
    Salim AA, Bakhtiar H, Ghoshal SK, Huyop F
    Opt Laser Technol, 2020 Oct;130:106331.
    PMID: 32457554 DOI: 10.1016/j.optlastec.2020.106331
    Biomedical values of organic natural cinnamon that are buried in their bulk counterpart can be exposed and customised via nanosizing. Based on this factor, a new type of spherical cinnamon nanoclusters (Cin-NCs) were synthesised using eco-friendly nanosecond pulse laser ablation in liquid (PLAL) approach. As-grown nontoxic Cin-NCs suspended in the citric acid of pH 4.5 (acted as organic solvent) were characterised thoroughly to evaluate their structural, optical and bactericidal properties. The effects of various laser fluences (LF) at the fixed wavelength (532 nm) on the physiochemical properties of these Cin-NCs were determined. The FTIR spectra of the Cin-NCs displayed the symmetric-asymmetric stretching of the functional groups attached to the heterocyclic/cinnamaldehyde compounds. The HR-TEM image of the optimum sample revealed the nucleation of the crystalline spherical Cin-NCs with a mean diameter of approximately 10 ± 0.3 nm and lattice fringe spacing around 0.14 nm. In addition, the inhibition zone diameter (IZD) and optical density (OD600) of the proposed Cin-NCs were measured to assess their antibacterial potency against the Staphylococcus aureus (IZD ≈ 24 mm) and Escherichia coli (IZD ≈ 25 mm) bacterial strains. The strong UV absorption (in the range of 269 and 310 nm) shown by these NCs was established to be useful for the antibacterial drug development and food treatment.
  8. Whole genome strategies and bioremediation insight into dehalogenase-producing bacteria
    Oyewusi HA, Wahab RA, Huyop F
    Mol Biol Rep, 2021 Mar;48(3):2687-2701.
    PMID: 33650078 DOI: 10.1007/s11033-021-06239-7
    An integral approach to decoding both culturable and uncultured microorganisms' metabolic activity involves the whole genome sequencing (WGS) of individual/complex microbial communities. WGS of culturable microbes, amplicon sequencing, metagenomics, and single-cell genome analysis are selective techniques integrating genetic information and biochemical mechanisms. These approaches transform microbial biotechnology into a quick and high-throughput culture-independent evaluation and exploit pollutant-degrading microbes. They are windows into enzyme regulatory bioremediation pathways (i.e., dehalogenase) and the complete bioremediation process of organohalide pollutants. While the genome sequencing technique is gaining the scientific community's interest, it is still in its infancy in the field of pollutant bioremediation. The techniques are becoming increasingly helpful in unraveling and predicting the enzyme structure and explore metabolic and biodegradation capabilities.
  9. Dehalogenase-producing halophiles and their potential role in bioremediation
    Oyewusi HA, Wahab RA, Huyop F
    Mar Pollut Bull, 2020 Nov;160:111603.
    PMID: 32919122 DOI: 10.1016/j.marpolbul.2020.111603
    This review aims to briefly describe the potential role of dehalogenase-producing halophilic bacteria in decontamination of organohalide pollutants. Hypersaline habitats pose challenges to life because of low water activity (water content) and is considered as the largest and ultimate sink for pollutants due to naturally and anthropogenic activities in which a substantial amount of ecological contaminants are organohalides. Several such environments appear to host and support substantial diversity of extremely halophilic and halotolerant bacteria as well as halophilic archaea. Biodegradation of several toxic inorganic and organic compounds in both aerobic and anaerobic conditions are carried out by halophilic microbes. Therefore, remediation of polluted marine/hypersaline environments are the main scorching issues in the field of biotechnology. Although many microbial species are reported as effective pollutants degrader, but little has been isolated from marine/hypersaline environments. Therefore, more novel microbial species with dehalogenase-producing ability are still desired.
  10. The mechanistic role of active site residues in non-stereo haloacid dehalogenase E (DehE)
    Zainal Abidin MH, Abd Halim KB, Huyop F, Tengku Abdul Hamid TH, Abdul Wahab R, Abdul Hamid AA
    J Mol Graph Model, 2019 07;90:219-225.
    PMID: 31103914 DOI: 10.1016/j.jmgm.2019.05.003
    Dehalogenase E (DehE) is a non-stereospecific enzyme produced by the soil bacterium, Rhizobium sp. RC1. Till now, the catalytic mechanism of DehE remains unclear although several literature concerning its structure and function are available. Since DehE is non-stereospecific, the enzyme was hypothesized to follow a 'direct attack mechanism' for the catalytic breakdown of a haloacid. For a molecular insight, the DehE modelled structure was docked in silico with the substrate 2-chloropropionic acid (2CP) in the active site. The ideal position of DehE residues that allowed a direct attack mechanism was then assessed via molecular dynamics (MD) simulation. It was revealed that the essential catalytic water was hydrogen bonded to the 'water-bearer', Asn114, at a relatively constant distance of ∼2.0 Å after 50 ns. The same water molecule was also closely sited to the catalytic Asp189 at an average distance of ∼2.0 Å, signifying the imperative role of the latter to initiate proton abstraction for water activation. This reaction was crucial to promote a direct attack on the α-carbon of 2CP to eject the halide ion. The water molecule was oriented favourably towards the α-carbon of 2CP at an angle of ∼75°, mirrored by the formation of stable enzyme-substrate orientations throughout the simulation. The data therefore substantiated that the degradation of a haloacid by DehE followed a 'direct attack mechanism'. Hence, this study offers valuable information into future advancements in the engineering of haloacid dehalogenases with improved activity and selectivity, as well as functionality in solvents other than water.
  11. Theoretical analyses on enantiospecificity of L-2-haloacid dehalogenase (DehL) from Rhizobium sp. RC1 towards 2-chloropropionic acid
    Adamu A, Abdul Wahab R, Aliyu F, Abdul Razak FI, Mienda BS, Shamsir MS, et al.
    J Mol Graph Model, 2019 11;92:131-139.
    PMID: 31352207 DOI: 10.1016/j.jmgm.2019.07.012
    Dehalogenases continue to garner interest of the scientific community due to their potential applications in bioremediation of halogen-contaminated environment and in synthesis of various industrially relevant products. Example of such enzymes is DehL, an L-2-haloacid dehalogenase (EC 3.8.1.2) from Rhizobium sp. RC1 that catalyses the specific cleavage of halide ion from L-2-halocarboxylic acids to produce the corresponding D-2-hydroxycarboxylic acids. Recently, the catalytic residues of DehL have been identified and its catalytic mechanism has been fully elucidated. However, the enantiospecificity determinants of the enzyme remain unclear. This information alongside a well-defined catalytic mechanism are required for rational engineering of DehL for substrate enantiospecificity. Therefore, using quantum mechanics/molecular mechanics and molecular mechanics Poisson-Boltzmann surface area calculations, the current study theoretically investigated the molecular basis of DehL enantiospecificity. The study found that R51L mutation cancelled out the dehalogenation activity of DehL towards it natural substrate, L-2-chloropropionate. The M48R mutation, however introduced a new activity towards D-2-chloropropionate, conveying the possibility of inverting the enantiospecificity of DehL from L-to d-enantiomer with a minimum of two simultaneous mutations. The findings presented here will play important role in the rational design of DehL dehalogenase for improving substrate utility.
  12. Raw oil palm frond leaves as cost-effective substrate for cellulase and xylanase productions by Trichoderma asperellum UC1 under solid-state fermentation
    Ezeilo UR, Lee CT, Huyop F, Zakaria II, Wahab RA
    J Environ Manage, 2019 Aug 01;243:206-217.
    PMID: 31096173 DOI: 10.1016/j.jenvman.2019.04.113
    Production of cellulases and xylanase by a novel Trichoderma asperellum UC1 (GenBank accession no. MF774876) under solid state fermentation (SSF) of raw oil palm frond leaves (OPFL) was optimized. Under optimum fermentation parameters (30 °C, 60-80% moisture content, 2.5 × 106 spores/g inoculum size) maximum CMCase, FPase, β-glucosidase and xylanase activity were recorded at 136.16 IU/g, 26.03 U/g, 130.09 IU/g and 255.01 U/g, respectively. Cellulases and xylanase were produced between a broad pH range of pH 6.0-12.0. The enzyme complex that comprised of four endo-β-1,4-xylanases and endoglucanases, alongside exoglucanase and β-glucosidase showed thermophilic and acidophilic characteristics at 50-60 °C and pH 3.0-4.0, respectively. Glucose (16.87 mg/g) and fructose (18.09 mg/g) were among the dominant sugar products from the in situ hydrolysis of OPFL, aside from cellobiose (105.92 mg/g) and xylose (1.08 mg/g). Thermal and pH stability tests revealed that enzymes CMCase, FPase, β-glucosidase and xylanase retained 50% residual activities for up to 15.18, 4.06, 17.47 and 15.16 h of incubation at 60 °C, as well as 64.59, 25.14, 68.59 and 19.20 h at pH 4.0, respectively. Based on the findings, it appeared that the unique polymeric structure of raw OPFL favored cellulases and xylanase productions.
  13. Haloadaptation: insights from comparative modeling studies between halotolerant and non-halotolerant dehalogenases
    Edbeib MF, Aksoy HM, Kaya Y, Wahab RA, Huyop F
    J Biomol Struct Dyn, 2020 Aug;38(12):3452-3461.
    PMID: 31422756 DOI: 10.1080/07391102.2019.1657498
    Halophiles are extremophilic microorganisms that grow optimally at high salt concentrations by producing a myriad of equally halotolerant enzymes. Structural haloadaptation of these enzymes adept to thriving under high-salt environments, though are not fully understood. Herein, the study attempts an in silico investigation to identify and comprehend the evolutionary structural adaptation of a halotolerant dehalogenase, DehHX (GenBank accession number: KR297065) of the halotolerant Pseudomonas halophila, over its non-halotolerant counterpart, DehMX1 (GenBank accession number KY129692) produced by Pseudomonas aeruginosa. GC content of the halotolerant DehHX DNA sequence was distinctively higher (58.9%) than the non-halotolerant dehalogenases (55% average GC). Its acidic residues, Asp and Glu were 8.27% and 12.06%, respectively, compared to an average 5.5% Asp and 7% Glu, in the latter; but lower contents of basic and hydrophobic residues in the DehHX. The secondary structure of DehHX interestingly revealed a lower incidence of α-helix forming regions (29%) and a higher percentage of coils (57%), compared to 49% and 29% in the non-halotolerant homologues, respectively. Simulation models showed the DehHX is stable under a highly saline environment (25% w/v) by adopting a highly negative-charged surface with a concomitant weakly interacting hydrophobic core. The study thus, established that a halotolerant dehalogenase undergoes notable evolutionary structural changes related to GC content over its non-halotolerant counterpart, in order to adapt and thrive under highly saline environments.Communicated by Ramaswamy H. Sarma.
  14. In silico mutation on a mutant lipase from Acinetobacter haemolyticus towards enhancing alkaline stability
    Anuar NFSK, Wahab RA, Huyop F, Halim KBA, Hamid AAA
    J Biomol Struct Dyn, 2020 Sep;38(15):4493-4507.
    PMID: 31630644 DOI: 10.1080/07391102.2019.1683074
    Alkaline-stable lipases are highly valuable biocatalysts that catalyze reactions under highly basic conditions. Herein, computational predictions of lipase from Acinetobacter haemolyticus and its mutant, Mut-LipKV1 was performed to identify functionally relevant mutations that enhance pH performance under increasing basicity. Mut-LipKV1 was constructed by in silico site directed mutagenesis of several outer loop acidic residues, aspartic acid (Asp) into basic ones, lysine (Lys) at positions 51, 122 and 247, followed by simulation under extreme pH conditions (pH 8.0-pH 12.0). The energy minimized Mut-LipKV1 model exhibited good quality as shown by PROCHECK, ERRAT and Verify3D data that corresponded to 79.2, 88.82 and 89.42% in comparison to 75.2, 86.15, and 95.19% in the wild-type. Electrostatic surface potentials and charge distributions of the Mut-LipKV1 model was more stable and better adapted to conditions of elevated pHs (pH 8.0 - 10.0). Mut-LipKV1 exhibited a mixture of neutral and positive surface charge distribution compared to the predominantly negative charge in the wild-type lipase at pH 8.0. Data of molecular dynamics simulations also supported the increased alkaline-stability of Mut-LipKV1, wherein the lipase was more stable at a higher pH 9.0 (RMSD = ∼0.3 nm, RMSF = ∼0.05-0.2 nm), over the optimal pH 8.0 of the wild-type lipase (RMSD = 0.3 nm, RMSF = 0.05-0.20 nm). Thus, the adaptive strategy of replacing surface aspartic acid to lysine in lipase was successful in yielding a more alkaline-stable Mut-LipKV1 under elevated basic conditions.Communicated by Ramaswamy H. Sarma.
  15. Biodegradation of 3-chloropropionic acid (3-CP) by Bacillus cereus WH2 and its in silico enzyme-substrate docking analysis
    Muslem WH, Edbeib MF, Aksoy HM, Kaya Y, Hamid AAA, Hood MHM, et al.
    J Biomol Struct Dyn, 2020 07;38(11):3432-3441.
    PMID: 31401940 DOI: 10.1080/07391102.2019.1655482
  16. Molecular docking and molecular dynamics simulation of Bacillus thuringiensis dehalogenase against haloacids, haloacetates and chlorpyrifos
    Oyewusi HA, Huyop F, Wahab RA
    J Biomol Struct Dyn, 2020 Oct 23.
    PMID: 33094694 DOI: 10.1080/07391102.2020.1835727
    The high dependency and surplus use of agrochemical products have liberated enormous quantities of toxic halogenated pollutants into the environment and threaten the well-being of humankind. Herein, this study performed molecular docking, molecular dynamic (MD) simulations, molecular mechanics-Poisson Boltzmann Surface Area (MM-PBSA) calculations on the DehH2 from Bacillus thuringiensis, to identify the order of which the enzyme degrades different substrates, haloacids, haloacetate and chlorpyrifos. The study discovered that the DehH2 favored the degradation of haloacids and haloacetates (-3.3 - 4.6 kcal/mol) and formed three hydrogen bonds with Asp125, Arg201 and Lys202. Despite the inconclusive molecular docking result, chlorpyrifos was consistently shown to be the least favored substrate of the DehH2 in MD simulations and MM-PBSA calculations. Results of MD simulations revealed the DehH2-haloacid- (RMSD 0.15 - 0.25 nm) and DehH2-haloacetates (RMSF 0.05 - 0.25 nm) were more stable, with the DehH2-L-2CP complex being the most stable while the least was the DehH2-chlorpyrifos (RMSD 0.295 nm; RMSF 0.05 - 0.59 nm). The Molecular Mechanics Poisson-Boltzmann Surface Area calculations showed the DehH2-L-2CP complex (-24.27 kcal/mol) having the lowest binding energy followed by DehH2-MCA (-22.78 kcal/mol), DehH2-D-2CP (-21.82 kcal/mol), DehH2-3CP (-21.11 kcal/mol), DehH2-2,2-DCP (-18.34 kcal/mol), DehH2-2,3-DCP (-8.34 kcal/mol), DehH2-TCA (-7.62 kcal/mol), while chlorpyrifos was unable to spontaneously bind to DehH2 (+127.16 kcal/mol). In a nutshell, the findings of this study offer valuable insights into the rational tailoring of the DehH2 for expanding its substrate specificity and catalytic activity in the near future.Communicated by Ramaswamy H. Sarma.
  17. Molecular docking and molecular dynamics simulations of a mutant Acinetobacter haemolyticus alkaline-stable lipase against tributyrin
    Anuar NFSK, Wahab RA, Huyop F, Amran SI, Hamid AAA, Halim KBA, et al.
    J Biomol Struct Dyn, 2021 Apr;39(6):2079-2091.
    PMID: 32174260 DOI: 10.1080/07391102.2020.1743364
    We previously reported on a mutant lipase KV1 (Mut-LipKV1) from Acinetobacter haemolyticus which optimal pH was raised from 8.0 to 11.0 after triple substitutions of surface aspartic acid (Asp) with lysine (Lys). Herein, this study further examined the Mut-LipKV1 by molecular docking, molecular dynamics (MD) simulations and molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) calculations to explore the structural requirements that participated in the effective binding of tributyrin and its catalytic triad (Ser165, Asp259 and His289) and identify detailed changes that occurred post mutation. Mut-LipKV1 bound favorably with tributyrin (-4.1 kcal/mol) and formed a single hydrogen bond with His289, at pH 9.0. Despite the incongruent docking analysis data, results of MD simulations showed configurations of both the tributyrin-Mut-LipKV1 (RMSD 0.3 nm; RMSF 0.05 - 0.3 nm) and the tributyrin-wildtype lipase KV1 (tributyrin-LipKV1) complexes (RMSD 0.35 nm; RMSF 0.05 - 0.4 nm) being comparably stable at pH 8.0. MM-PBSA analysis indicated that van der Waals interactions made the most contribution during the molecular binding process, with the Mut-LipKV1-tributyrin complex (-44.04 kcal/mol) showing relatively lower binding energy than LipKV1-tributyrin (-43.83 kcal/mol), at pH 12.0. All tributyrin-Mut-LipKV1 complexes displayed improved binding free energies over a broader pH range from 8.0 - 12.0, as compared to LipKV1-tributyrin. Future empirical works are thus, important to validate the improved alkaline-stability of Mut-LipKV1. In a nutshell, our research offered a considerable insight for further improving the alkaline tolerance of lipases.Communicated by Ramaswamy H. Sarma.
  18. Multi-template homology-based structural model of L-2-haloacid dehalogenase (DehL) from Rhizobium sp. RC1
    Adamu A, Shamsir MS, Wahab RA, Parvizpour S, Huyop F
    J Biomol Struct Dyn, 2017 Nov;35(15):3285-3296.
    PMID: 27800712 DOI: 10.1080/07391102.2016.1254115
    Dehalogenases are of high interest due to their potential applications in bioremediation and in synthesis of various industrial products. DehL is an L-2-haloacid dehalogenase (EC 3.8.1.2) that catalyses the cleavage of halide ion from L-2-halocarboxylic acid to produce D-2-hydroxycarboxylic acid. Although DehL utilises the same substrates as the other L-2-haloacid dehalogenases, its deduced amino acid sequence is substantially different (<25%) from those of the rest L-2-haloacid dehalogenases. To date, the 3D structure of DehL is not available. This limits the detailed understanding of the enzyme's reaction mechanism. The present work predicted the first homology-based model of DehL and defined its active site. The monomeric unit of the DehL constitutes α/β structure that is organised into two distinct structural domains: main and subdomains. Despite the sequence disparity between the DehL and other L-2-haloacid dehalogenases, its structural model share similar fold as the experimentally solved L-DEX and DehlB structures. The findings of the present work will play a crucial role in elucidating the molecular details of the DehL functional mechanism.
  19. In silico and empirical approaches toward understanding the structural adaptation of the alkaline-stable lipase KV1 from Acinetobacter haemolyticus
    Batumalaie K, Edbeib MF, Mahat NA, Huyop F, Wahab RA
    J Biomol Struct Dyn, 2018 Sep;36(12):3077-3093.
    PMID: 28884626 DOI: 10.1080/07391102.2017.1377635
    Interests in Acinetobacter haemolyticus lipases are showing an increasing trend concomitant with growth of the enzyme industry and the widening search for novel enzymes and applications. Here, we present a structural model that reveals the key catalytic residues of lipase KV1 from A. haemolyticus. Homology modeling of the lipase structure was based on the structure of a carboxylesterase from the archaeon Archaeoglobus fulgidus as the template, which has a sequence that is 58% identical to that of lipase KV1. The lipase KV1 model is comprised of a single compact domain consisting of seven parallel and one anti-parallel β-strand surrounded by nine α-helices. Three structurally conserved active-site residues, Ser165, Asp259, and His289, and a tunnel through which substrates access the binding site were identified. Docking of the substrates tributyrin and palmitic acid into the pH 8 modeled lipase KV1 active sites revealed an aromatic platform responsible for the substrate recognition and preference toward tributyrin. The resulting binding modes from the docking simulation correlated well with the experimentally determined hydrolysis pattern, for which pH 8 and tributyrin being the optimum pH and preferred substrate. The results reported herein provide useful insights into future structure-based tailoring of lipase KV1 to modulate its catalytic activity.
  20. In silico assessment of dehalogenase from Bacillus thuringiensis H2 in relation to its salinity-stability and pollutants degradation
    Oyewusi HA, Huyop F, Wahab RA, Hamid AAA
    J Biomol Struct Dyn, 2022;40(19):9332-9346.
    PMID: 34014147 DOI: 10.1080/07391102.2021.1927846
    Increased scientific interest has led to the rise in biotechnological uses of halophilic and halotolerant microbes for hypersaline wastewater bioremediation. Hence, this study performed molecular docking, molecular dynamic (MD) simulations, and validation by Molecular Mechanic Poisson-Boltzmann Surface Area (MM-PBSA) calculations on the DehH2 from Bacillus thuringiensis H2. We aimed to identify the interactions of DehH2 with substrates haloacids, haloacetates, and chlorpyrifos under extreme salinity (35% NaCl). MD simulations revealed that DehH2 preferentially degraded haloacids and haloacetates (-6.3 to -4.7 kcal/mol) by forming three or four hydrogen bonds to the catalytic triad, Asp125, Arg201, and Lys202. Conversely, chlorpyrifos was the least preferred substrate in both MD simulations and MM-PBSA calculations. MD simulation results ranked the DehH2-L-2CP complex (RMSD □0.125-0.23 nm) as the most stable while the least was the DehH2-chlorpyrifos complex (RMSD 0.32 nm; RMSF 0.0 - 0.29). The order of stability was as follows: DehH2-L-2CP > DehH2-MCA > DehH2-D-2CP > DehH2-3CP > DehH2-2,2-DCP > DehH2-2,3-DCP > DehH2-TCA > DehH2-chlorpyrifos. The MM-PBSA calculations further affirmed the DehH2-L-2CP complex's highest stability with the lowest binding energy of -45.14 kcal/mol, followed closely by DehH2-MCA (-41.21 kcal/mol), DehH2-D-2CP (-31.59 kcal/mol), DehH2-3CP (-30.75 kcal/mol), DehH2-2,2- DCP (-29.72 kcal/mol), DehH2-2,3-DCP (-22.20 kcal/mol) and DehH2-TCA (-18.46 kcal/mol). The positive binding energy of the DehH2-chlorpyrifos complex (+180.57 kcal/mol) proved the enzyme's non-preference for the substrate. The results ultimately illustrated the unique specificity of the DehH2 to degrade the above-said pollutants under a hypersaline condition.Communicated by Ramaswamy H. Sarma.
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