Displaying publications 1 - 20 of 38 in total

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  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. The first ITS2 sequence data set of eDNA from honey of Malaysian giant honeybees (Apis dorsata) and stingless bees (Heterotrigona itama) reveals plant species diversity
    Huda N, Ullah S, Wahab RA, Lani MN, Daud NHA, Shariff AHM, et al.
    BMC Res Notes, 2023 Sep 12;16(1):211.
    PMID: 37700361 DOI: 10.1186/s13104-023-06495-9
    OBJECTIVES: Pollen is a useful tool for identifying the provenance and complex ecosystems surrounding honey production in Malaysian forests. As native key pollinators in Malaysia, Apis dorsata and Heterotrigona itama forage on various plant/pollen species to collect honey. This study aims to generate a dataset that uncovers the presence of these plant/pollen species and their relative abundance in the honey of A. dorsata and H. itama. The information gathered from this study can be used to determine the geographical and botanical origin and authenticity of the honey produced by these two species.

    RESULTS: Sequence data were obtained for both A. dorsata and H. itama. The raw sequence data for A. dorsata was 5 Mb, which was assembled into 5 contigs with a size of 6,098,728 bp, an N50 of 15,534, and a GC average of 57.42. Similarly, the raw sequence data for H. itama was 6.3 Mb, which was assembled into 11 contigs with a size of 7,642,048 bp, an N50 of 17,180, and a GC average of 55.38. In the honey sample of A. dorsata, we identified five different plant/pollen species, with only one of the five species exhibiting a relative abundance of less than 1%. For H. itama, we identified seven different plant/pollen species, with only three of the species exhibiting a relative abundance of less than 1%. All of the identified plant species were native to Peninsular Malaysia, especially the East Coast area of Terengganu.

    DATA DESCRIPTION: Our data offers valuable insights into honey's geographical and botanical origin and authenticity. Metagenomic studies could help identify the plant species that honeybees forage and provide preliminary data for researchers studying the biological development of A. dorsata and H. itama. The identification of various flowers from the eDNA of honey that are known for their medicinal properties could aid in regional honey with accurate product origin labeling, which is crucial for guaranteeing product authenticity to consumers.

  6. 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.
  7. Fulltext Structure prediction, molecular dynamics simulation and docking studies of D-specific dehalogenase from Rhizobium sp. RC1
    Sudi IY, Wong EL, Joyce-Tan KH, Shamsir MS, Jamaluddin H, Huyop F
    Int J Mol Sci, 2012;13(12):15724-54.
    PMID: 23443090 DOI: 10.3390/ijms131215724
    Currently, there is no three-dimensional structure of D-specific dehalogenase (DehD) in the protein database. We modeled DehD using ab initio technique, performed molecular dynamics (MD) simulation and docking of D-2-chloropropionate (D-2CP), D-2-bromopropionate (D-2BP), monochloroacetate (MCA), monobromoacetate (MBA), 2,2-dichloropropionate (2,2-DCP), d,l-2,3-dichloropropionate (d,l-2,3-DCP), and 3-chloropropionate (3-CP) into the DehD active site. The sequences of DehD and D-2-haloacid dehalogenase (HadD) from Pseudomonas putida AJ1 have 15% sequence similarity. The model had 80% of the amino acid residues in the most favored region when compared to the crystal structure of DehI from Pseudomonas putida PP3. Docking analysis revealed that Arg107, Arg134 and Tyr135 interacted with D-2CP, and Glu20 activated the water molecule for hydrolytic dehalogenation. Single residue substitutions at 25-30 °C showed that polar residues of DehD were stable when substituted with nonpolar residues and showed a decrease in activity within the same temperature range. The molecular dynamics simulation of DehD and its variants showed that in R134A variant, Arg107 interacted with D-2CP, while in Y135A, Gln221 and Arg231 interacted with D-2CP. It is our emphatic belief that the new model will be useful for the rational design of DehDs with enhanced potentials.
  8. 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.
  9. Prominent bactericidal characteristics of silver-copper nanocomposites produced via pulse laser ablation
    Alhajj M, Aziz MSA, Huyop F, Salim AA, Sharma S, Ghoshal SK
    Biomater Adv, 2022 Nov;142:213136.
    PMID: 36206587 DOI: 10.1016/j.bioadv.2022.213136
    This paper reports the characterization and antibacterial performance evaluation of some spherical and stable crystalline silver (Ag)/copper (Cu) nanocomposites (Ag-CuNCs) prepared in deionized water (DIW) using pulse laser ablation in liquid (PLAL) method. The influence of various laser fluences (LFs) on the structural, morphological, optical and antibacterial properties of these NCs were determined. The UV-Vis absorbance of these NCs at 403 nm and 595 nm was gradually increased accompanied by a blue shift. XRD patterns disclosed the nucleation of highly crystalline Ag-CuNCs with their face centered cubic lattice structure. TEM images showed the existence of spherical NCs with size range of 3-20 nm and lattice fringe spacing of approximately 0.145 nm. EDX profiles of Ag-CuNCs indicated their high purity. The antibacterial effectiveness of the Ag-CuNCs was evaluated by the inhibition zone diameter (IZD) and optical density (OD600) tests against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The proposed NCs revealed the IZD values in the range of 22-26 mm and 20-25 mm when tested against E. coli and S. aureus bacteria, respectively. The Ag-CuNCs prepared at LF of 14.15 J/cm2 revealed the best bactericidal activity. It is established that by controlling the laser fluence the bactericidal effectiveness of the Ag-CuNCs can be tuned.
  10. 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.
  11. 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.
  12. 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.
  13. 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.
  14. Interactions of non-natural halogenated substrates with D-specific dehalogenase (DehD) mutants using in silico studies
    Sudi IY, Shamsir MS, Jamaluddin H, Wahab RA, Huyop F
    Biotechnology, biotechnological equipment, 2014 Sep 03;28(5):949-957.
    PMID: 26019583
    The D-2-haloacid dehalogenase of D-specific dehalogenase (DehD) from Rhizobium sp. RC1 catalyses the hydrolytic dehalogenation of D-haloalkanoic acids, inverting the substrate-product configuration and thereby forming the corresponding L-hydroxyalkanoic acids. Our investigations were focused on DehD mutants: R134A and Y135A. We examined the possible interactions between these mutants with haloalkanoic acids and characterized the key catalytic residues in the wild-type dehalogenase, to design dehalogenase enzyme(s) with improved potential for dehalogenation of a wider range of substrates. Three natural substrates of wild-type DehD, specifically, monochloroacetate, monobromoacetate and D,L-2,3-dichloropropionate, and eight other non-natural haloalkanoic acids substrates of DehD, namely, L-2-chloropropionate; L-2-bromopropionate; 2,2-dichloropropionate; dichloroacetate; dibromoacetate; trichloroacetate; tribromoacetate; and 3-chloropropionate, were docked into the active site of the DehD mutants R134A and Y135A, which produced altered catalytic functions. The mutants interacted strongly with substrates that wild-type DehD does not interact with or degrade. The interaction was particularly enhanced with 3-chloropropionate, in addition to monobromoacetate, monochloroacetate and D,L-2,3-dichloropropionate. In summary, DehD variants R134A and Y135A demonstrated increased propensity for binding haloalkanoic acid and were non-stereospecific towards halogenated substrates. The improved characteristics in these mutants suggest that their functionality could be further exploited and harnessed in bioremediations and biotechnological applications.
  15. Insights into the stereospecificity of the d-specific dehalogenase from Rhizobium sp. RC1 toward d- and l-2-chloropropionate
    Sudi IY, Hamid AA, Shamsir MS, Jamaluddin H, Wahab RA, Huyop F
    Biotechnology, biotechnological equipment, 2014 Jul 04;28(4):608-615.
    PMID: 26740767
    Halogenated compounds are recalcitrant environmental pollutants prevalent in agricultural fields, waste waters and industrial by-products, but they can be degraded by dehalogenase-containing microbes. Notably, 2-haloalkanoic acid dehalogenases are employed to resolve optically active chloropropionates, as exemplified by the d-specific dehalogenase from Rhizobium sp. RCI (DehD), which acts on d-2-chloropropionate but not on its l-enantiomer. The catalytic residues of this dehalogenase responsible for its affinity toward d-2-chloropropionate have not been experimentally determined, although its three-dimensional crystal structure has been solved. For this study, we performed in silico docking and molecular dynamic simulations of complexes formed by this dehalogenase and d- or l-2-chloropropionate. Arg134 of the enzyme plays the key role in the stereospecific binding and Arg16 is in a position that would allow it to activate a water molecule for hydrolytic attack on the d-2-chloropropionate chiral carbon for release of the halide ion to yield l-2-hydroxypropionate. We propose that within the DehD active site, the NH group of Arg134 can form a hydrogen bond with the carboxylate of d-2-chloropropionate with a strength of ∼4 kcal/mol that may act as an acid-base catalyst, whereas, when l-2-chloropropionate is present, this bond cannot be formed. The significance of the present work is vital for rational design of this dehalogenase in order to confirm the involvement of Arg16 and Arg134 residues implicated in hydrolysis and binding of d-2-chloropropionate in the active site of d-specific dehalogenase from Rhizobium sp. RC1.
  16. 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.
  17. 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.
  18. 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.
  19. In silico analysis of a putative dehalogenase from the genome of halophilic bacterium Halomonas smyrnensis AAD6T
    Oyewusi HA, Akinyede KA, Abdul Wahab R, Huyop F
    J Biomol Struct Dyn, 2023 Jan;41(1):319-335.
    PMID: 34854349 DOI: 10.1080/07391102.2021.2006085
    Microbial-assisted removal of natural or synthetic pollutants is the prevailing green, low-cost technology to treat polluted environments. However, the challenge with enzyme-assisted bioremediation is the laborious nature of dehalogenase-producing microorganisms' bioprospecting. This bottleneck could be circumvented by in-silico analysis of certain microorganisms' whole-genome sequences to predict their protein functions and enzyme versatility for improved biotechnological applications. Herein, this study performed structural analysis on a dehalogenase (DehHsAAD6) from the genome of Halomonas smyrnensis AAD6 by molecular docking and molecular dynamic (MD) simulations. Other bioinformatics tools were also employed to identify substrate preference (haloacids and haloacetates) of the DehHsAAD6. The DehHsAAD6 preferentially degraded haloacids and haloacetates (-3.2-4.8 kcal/mol) and which formed three hydrogen bonds with Tyr12, Lys46, and Asp182. MD simulations data revealed the higher stability of DehHsAAD6-haloacid- (RMSD 0.22-0.3 nm) and DehHsAAD6-haloacetates (RMSF 0.05-0.14 nm) complexes, with the DehHsAAD6-L-2CP complex being the most stable. The detail of molecular docking calculations ranked complexes with the lowest binding free energies as: DehHsAAD6-L-2CP complex (-4.8 kcal/mol) = DehHsAAD6-MCA (-4.8 kcal/mol) < DehHsAAD6-TCA (-4.5 kcal/mol) < DehHsAAD6-2,3-DCP (-4.1 kcal/mol) < DehHsAAD6-D-2CP (-3.9 kcal/mol) < DehHsAAD6-2,2-DCP (-3.5 kcal/mol) < DehHsAAD6-3CP (-3.2 kcal/mol). In a nutshell, the study findings offer valuable perceptions into the elucidation of possible reaction mechanisms of dehalogenases for extended substrate specificity and higher catalytic activity.Communicated by Ramaswamy H. Sarma.
  20. Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1
    Hamid AA, Hamid TH, Wahab RA, Huyop F
    J Basic Microbiol, 2015 Mar;55(3):324-30.
    PMID: 25727054 DOI: 10.1002/jobm.201570031
    The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties.
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