Displaying publications 1 - 20 of 137 in total

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  1. Bacteriocin release protein-mediated secretory expression of recombinant chalcone synthase in Escherichia coli
    Zakaria II, Rahman RN, Salleh AB, Basri M
    Appl Biochem Biotechnol, 2011 Sep;165(2):737-47.
    PMID: 21633820 DOI: 10.1007/s12010-011-9292-1
    Flavonoids are secondary metabolites synthesized by plants shown to exhibit health benefits such as anti-inflammatory, antioxidant, and anti-tumor effects. Thus, due to the importance of this compound, several enzymes involved in the flavonoid pathway have been cloned and characterized in Escherichia coli. However, the formation of inclusion bodies has become a major disadvantage of this approach. As an alternative, chalcone synthase from Physcomitrella patens was secreted into the medium using a bacteriocin release protein expression vector. Secretion of P. patens chalcone synthase into the culture media was achieved by co-expression with a psW1 plasmid encoding bacteriocin release protein in E. coli Tuner (DE3) plysS. The optimized conditions, which include the incubation of cells for 20 h with 40 ng/ml mitomycin C at OD(600) induction time of 0.5 was found to be the best condition for chalcone synthase secretion.
  2. Kinetic behaviour of free lipase and mica-based immobilized lipase catalyzing the synthesis of sugar esters
    Zaidan UH, Abdul Rahman MB, Othman SS, Basri M, Abdulmalek E, Rahman RN, et al.
    Biosci Biotechnol Biochem, 2011;75(8):1446-50.
    PMID: 21821960
    The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K(m) and V(max), were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K(m) values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V(max,app)>V(max)). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.
  3. Fulltext Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati
    Yaacob N, Mohamad Ali MS, Salleh AB, Abdul Rahman NA
    PeerJ, 2016;4:e1751.
    PMID: 26989608 DOI: 10.7717/peerj.1751
    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2 expression, showing the highest expression when glucose was depleted and ethanol-acetic acid was increased. Meanwhile, S. cerevisiae showed a constitutive ADH2 expression throughout the fermentation process. Discussion. ADH2 expression in L. fermentati may be subjected to changes in the presence of non-fermentative carbon source. The nucleotide sequence showed that ADH2 transcription could be influenced by other transcription genes of glycolysis oriented due to the lack of specific activation sites for Adr1. Our study suggests that if Adr1 is not capable of promoting LfeADH2 activation, the transcription can be controlled by Rap1 and Sp1 due to their inherent roles. Therefore in future, it is interesting to observe ADH2 gene being highly regulated by these potential transcription factors and functioned as a promoter for yeast under high volume of ethanol and organic acids.
  4. Toluene promotes lid 2 interfacial activation of cold active solvent tolerant lipase from Pseudomonas fluorescens strain AMS8
    Yaacob N, Mohamad Ali MS, Salleh AB, Rahman RNZRA, Leow ATC
    J Mol Graph Model, 2016 07;68:224-235.
    PMID: 27474867 DOI: 10.1016/j.jmgm.2016.07.003
    The utilization of cold active lipases in organic solvents proves an excellent approach for chiral synthesis and modification of fats and oil due to the inherent flexibility of lipases under low water conditions. In order to verify whether this lipase can function as a valuable synthetic catalyst, the mechanism concerning activation of the lid and interacting solvent residues in the presence of organic solvent must be well understood. A new alkaline cold-adapted lipase, AMS8, from Pseudomonas fluorescens was studied for its structural adaptation and flexibility prior to its exposure to non-polar, polar aprotic and protic solvents. Solvents such as ethanol, toluene, DMSO and 2-propanol showed to have good interactions with active sites. Asparagine (Asn) and tyrosine (Tyr) were key residues attracted to solvents because they could form hydrogen bonds. Unlike in other solvents, Phe-18, Tyr-236 and Tyr-318 were predicted to have aromatic-aromatic side-chain interactions with toluene. Non-polar solvent also was found to possess highest energy binding compared to polar solvents. Due to this circumstance, the interaction of toluene and AMS8 lipase was primarily based on hydrophobicity and molecular recognition. The molecular dynamic simulation showed that lid 2 (residues 148-167) was very flexible in toluene and Ca(2+). As a result, lid 2 moves away from the catalytic areas, leaving an opening for better substrate accessibility which promotes protein activation. Only a single lid (lid 2) showed the movement following interactions with toluene, although AMS8 lipase displayed double lids. The secondary conformation of AMS8 lipase that was affected by toluene observed a reduction of helical strands and increased coil structure. Overall, this work shows that cold active lipase, AMS8 exhibits distinguish interfacial activation and stability in the presence of polar and non-polar solvents.
  5. Effects of Lid 1 Mutagenesis on Lid Displacement, Catalytic Performances and Thermostability of Cold-active Pseudomonas AMS8 Lipase in Toluene
    Yaacob N, Ahmad Kamarudin NH, Leow ATC, Salleh AB, Rahman RNZRA, Ali MSM
    Comput Struct Biotechnol J, 2019;17:215-228.
    PMID: 30828413 DOI: 10.1016/j.csbj.2019.01.005
    Pseudomonas fluorescens AMS8 lipase lid 1 structure is rigid and holds unclear roles due to the absence of solvent-interactions. Lid 1 region was stabilized by 17 hydrogen bond linkages and displayed lower mean hydrophobicity (0.596) compared to MIS38 lipase. Mutating lid 1 residues, Thr-52 and Gly-55 to aromatic hydrophobic-polar tyrosine would churned more side-chain interactions between lid 1 and water or toluene. This study revealed that T52Y leads G55Y and its recombinant towards achieving higher solvent-accessible surface area and longer half-life at 25 to 37 °C in 0.5% (v/v) toluene. T52Y also exhibited better substrate affinity with long-chain carbon substrate in aqueous media. The affinity for pNP palmitate, laurate and caprylate increased in 0.5% (v/v) toluene in recombinant AMS8, but the affinity in similar substrates was substantially declined in lid 1 mutated lipases. Regarding enzyme efficiency, the recombinant AMS8 lipase displayed highest value of kcat/Km in 0.5% (v/v) toluene, mainly with pNPC. In both hydrolysis reactions with 0% and 0.5% (v/v) toluene, the enzyme efficiency of G55Y was found higher than T52Y for pNPL and pNPP. At 0.5% (v/v) toluene, both mutants showed reductions in activation energy and enthalpy values as temperature increased from 25 to 35 °C, displaying better catalytic functions. Only T52Y exhibited increase in entropy values at 0.5% (v/v) toluene indicating structure stability. As a conclusion, Thr-52 and Gly-55 are important residues for lid 1 stability as their existence helps to retain the geometrical structure of alpha-helix and connecting hinge.
  6. Fulltext The Role of Solvent-Accessible Leu-208 of Cold-Active Pseudomonas fluorescens Strain AMS8 Lipase in Interfacial Activation, Substrate Accessibility and Low-Molecular Weight Esterification in the Presence of Toluene
    Yaacob N, Ahmad Kamarudin NH, Leow ATC, Salleh AB, Raja Abd Rahman RNZ, Mohamad Ali MS
    Molecules, 2017 Aug 12;22(8).
    PMID: 28805665 DOI: 10.3390/molecules22081312
    The alkaline cold-active lipase from Pseudomonas fluorescens AMS8 undergoes major structural changes when reacted with hydrophobic organic solvents. In toluene, the AMS8 lipase catalytic region is exposed by the moving hydrophobic lid 2 (Glu-148 to Gly-167). Solvent-accessible surface area analysis revealed that Leu-208, which is located next to the nucleophilic Ser-207 has a focal function in influencing substrate accessibility and flexibility of the catalytic pocket. Based on molecular dynamic simulations, it was found that Leu-208 strongly facilitates the lid 2 opening via its side-chain. The KM and Kcat/KM of L208A mutant were substrate dependent as it preferred a smaller-chain ester (pNP-caprylate) as compared to medium (pNP-laurate) or long-chain (pNP-palmitate) esters. In esterification of ethyl hexanoate, L208A promotes a higher ester conversion rate at 20 °C but not at 30 °C, as a 27% decline was observed. Interestingly, the wild-type (WT) lipase's conversion rate was found to increase with a higher temperature. WT lipase AMS8 esterification was higher in toluene as compared to L208A. Hence, the results showed that Leu-208 of AMS8 lipase plays an important role in steering a broad range of substrates into its active site region by regulating the flexibility of this region. Leu-208 is therefore predicted to be crucial for its role in interfacial activation and catalysis in toluene.
  7. Characterisation and molecular dynamic simulations of J15 asparaginase from Photobacterium sp. strain J15
    Yaacob MA, Hasan WA, Ali MS, Rahman RN, Salleh AB, Basri M, et al.
    Acta Biochim. Pol., 2014;61(4):745-52.
    PMID: 25337608
    Genome mining revealed a 1011 nucleotide-long fragment encoding a type I L-asparaginase (J15 asparaginase) from the halo-tolerant Photobacterium sp. strain J15. The gene was overexpressed in pET-32b (+) vector in E. coli strain Rosetta-gami B (DE3) pLysS and purified using two-step chromatographic methods: Ni(2+)-Sepharose affinity chromatography and Q-Sepharose anion exchange chromatography. The final specific activity and yield of the enzyme achieved from these steps were 20 U/mg and 49.2%, respectively. The functional dimeric form of J15-asparaginase was characterised with a molecular weight of ~70 kDa. The optimum temperature and pH were 25°C and pH 7.0, respectively. This protein was stable in the presence of 1 mM Ni(2+) and Mg(2+), but it was inhibited by Mn(2+), Fe(3+) and Zn(2+) at the same concentration. J15 asparaginase actively hydrolysed its native substrate, l-asparagine, but had low activity towards l-glutamine. The melting temperature of J15 asparaginase was ~51°C, which was determined using denatured protein analysis of CD spectra. The Km, Kcat, Kcat/Km of J15 asparaginase were 0.76 mM, 3.2 s(-1), and 4.21 s(-1) mM(-1), respectively. Conformational changes of the J15 asparaginase 3D structure at different temperatures (25°C, 45°C, and 65°C) were analysed using Molecular Dynamic simulations. From the analysis, residues Tyr₂₄ , His₂₂, Gly₂₃, Val₂₅ and Pro₂₆ may be directly involved in the 'open' and 'closed' lid-loop conformation, facilitating the conversion of substrates during enzymatic reactions. The properties of J15 asparaginase, which can work at physiological pH and has low glutaminase activity, suggest that this could be a good candidate for reducing toxic effects during cancer treatment.
  8. Organic-solvent stability of elastase strain K overexpressed in an Escherichia-Pseudomonas expression system
    Wong CF, Salleh AB, Basri M, Abd Rahman RN
    Biotechnol Appl Biochem, 2010 Sep;57(1):1-7.
    PMID: 20726840 DOI: 10.1042/BA20100224
    The structural gene of elastase strain K (elastase from Pseudomonas aeruginosa strain K), namely HindIII1500PstI, was successfully sequenced to contain 1497 bp. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consists of 301 amino acids, with a molecular mass of 33.1 kDa, and contains a conserved motif HEXXH, zinc ligands and residues involved in the catalysis of elastase strain K. The structural gene was successfully cloned to a shuttle vector, pUCP19, and transformed into Escherichia coli strains TOP10, KRX, JM109 and Tuner™ pLacI as well as P. aeruginosa strains PA01 (A.T.C.C. 47085) and S5, with detection of significant protein expression. Overexpression was detected from transformants KRX/pUCP19/HindIII1500PstI of E. coli and PA01/pUCP19/HindIII1500PstI of P. aeruginosa, with increases in elastolytic activity to 13.83- and 5.04-fold respectively relative to their controls. In addition, recombinant elastase strain K showed considerable stability towards numerous organic solvents such as methanol, ethanol, acetone, toluene, undecan-1-ol and n-dodecane, which typically pose a detrimental effect on enzymes; our finding provides further information to support the potential application of the enzyme in synthetic industries, particularly peptide synthesis.
  9. Fulltext Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa
    Wong CF, Rahman RNZRA, Basri M, Salleh AB
    Iran J Biotechnol, 2017;15(3):194-200.
    PMID: 29845069 DOI: 10.15171/ijb.1524
    Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.
  10. Enzymatic production of a solvent-free menthyl butyrate via response surface methodology catalyzed by a novel thermostable lipase from Geobacillus zalihae
    Wahab RA, Basri M, Rahman RN, Salleh AB, Rahman MB, Chaibakhsh N, et al.
    Biotechnology, biotechnological equipment, 2014 Nov 02;28(6):1065-1072.
    PMID: 26740782
    Most substrate for esterification has the inherent problem of low miscibility which requires addition of solvents into the reaction media. In this contribution, we would like to present an alternative and feasible option for an efficient solvent-free synthesis of menthyl butyrate using a novel thermostable crude T1 lipase. We investigated the effects of incubation time, temperature, enzyme loading and substrate molar ratio and determined the optimum conditions. The high conversion of menthyl butyrate catalyzed by crude T1 lipase in a solvent-free system is greatly affected by temperature and time of the reaction media. The highest yield of menthyl butyrate was 99.3% under optimized conditions of 60 °C, incubation time of 13.15 h, 2.53 mg, 0.43% (w/w) enzyme to substrate ratio and at molar ratio of butyric anhydride/menthol 2.7:1. Hence, the investigation revealed that the thermostable crude T1 lipase successfully catalyzed the high-yield production of menthyl butyrate in a solvent-free system. The finding suggests that the crude T1 lipase was a promising alternative to overcome shortcomings associated with solvent-assisted enzymatic reactions.
  11. Fulltext Combination of oxyanion Gln114 mutation and medium engineering to influence the enantioselectivity of thermophilic lipase from Geobacillus zalihae
    Wahab RA, Basri M, Rahman MB, Rahman RN, Salleh AB, Leow TC
    Int J Mol Sci, 2012;13(9):11666-80.
    PMID: 23109876 DOI: 10.3390/ijms130911666
    The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.
  12. Manipulation of the conformation and enzymatic properties of T1 lipase by site-directed mutagenesis of the protein core
    Wahab RA, Basri M, Rahman RN, Salleh AB, Rahman MB, Chor LT
    Appl Biochem Biotechnol, 2012 Jun;167(3):612-20.
    PMID: 22581079 DOI: 10.1007/s12010-012-9728-2
    In silico and experimental investigations were conducted to explore the effects of substituting hydrophobic residues, Val, Met, Leu, Ile, Trp, and Phe into Gln 114 of T1 lipase. The in silico investigations accurately predicted the enzymatic characteristics of the mutants in the experimental studies and provided rationalization for some of the experimental observations. Substitution with Leu successfully improved the conformational stability and enzymatic characteristics of T1 lipase. However, replacement of Gln114 with Trp negatively affected T1 lipase and resulted in the largest disruption of protein stability, diminished lipase activity and inferior enzymatic characteristics. These results suggested that the substitution of a larger residue in a densely packed area of the protein core can have considerable effects on the structure and function of an enzyme. This is especially true when the residue is next to the catalytic serine as demonstrated with the Phe and Trp mutation.
  13. Fulltext Discovery of a new class of inhibitors for the protein arginine deiminase type 4 (PAD4) by structure-based virtual screening
    Teo CY, Shave S, Chor AL, Salleh AB, Rahman MB, Walkinshaw MD, et al.
    BMC Bioinformatics, 2012;13 Suppl 17:S4.
    PMID: 23282142 DOI: 10.1186/1471-2105-13-S17-S4
    BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiology. Anticitrullinated protein autoantibody has been documented as a highly specific autoantibody associated with RA. Protein arginine deiminase type 4 (PAD4) is the enzyme responsible for catalyzing the conversion of peptidylarginine into peptidylcitrulline. PAD4 is a new therapeutic target for RA treatment. In order to search for inhibitors of PAD4, structure-based virtual screening was performed using LIDAEUS (Ligand discovery at Edinburgh university). Potential inhibitors were screened experimentally by inhibition assays.

    RESULTS: Twenty two of the top-ranked water-soluble compounds were selected for inhibitory screening against PAD4. Three compounds showed significant inhibition of PAD4 and their IC50 values were investigated. The structures of the three compounds show no resemblance with previously discovered PAD4 inhibitors, nor with existing drugs for RA treatment.

    CONCLUSION: Three compounds were discovered as potential inhibitors of PAD4 by virtual screening. The compounds are commercially available and can be used as scaffolds to design more potent inhibitors against PAD4.

  14. Novel furan-containing peptide-based inhibitors of protein arginine deiminase type IV (PAD4)
    Teo CY, Tejo BA, Leow ATC, Salleh AB, Abdul Rahman MB
    Chem Biol Drug Des, 2017 Dec;90(6):1134-1146.
    PMID: 28581157 DOI: 10.1111/cbdd.13033
    Protein arginine deiminase type IV (PAD4) is responsible for the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullinated protein is the autoantigen in rheumatoid arthritis, and therefore, PAD4 is currently a promising therapeutic target for the disease. Recently, we reported the importance of the furan ring in the structure of PAD4 inhibitors. In this study, the furan ring was incorporated into peptides to act as the "warhead" of the inhibitors for PAD4. IC50 studies showed that the furan-containing peptide-based inhibitors were able to inhibit PAD4 to a better extent than the furan-containing small molecules that were previously reported. The best peptide-based inhibitor inhibited PAD4 reversibly and competitively with an IC50 value of 243.2 ± 2.4 μm. NMR spectroscopy and NMR-restrained molecular dynamic simulations revealed that the peptide-based inhibitor had a random structure. Molecular docking studies showed that the peptide-based inhibitor entered the binding site and interacted with the essential amino acids involved in the catalytic activity. The peptide-based inhibitor could be further developed into a therapeutic drug for rheumatoid arthritis.
  15. Fulltext A potential tocopherol acetate loaded palm oil esters-in-water nanoemulsions for nanocosmeceuticals
    Teo BS, Basri M, Zakaria MR, Salleh AB, Rahman RN, Rahman MB
    J Nanobiotechnology, 2010;8:4.
    PMID: 20178581 DOI: 10.1186/1477-3155-8-4
    Cosmeceuticals are cosmetic-pharmaceutical hybrids intended to enhance health and beauty of the skin. Nanocosmeceuticals use nano-sized system for the delivery of active ingredients to the targeted cells for better penetration. In this work, nanoemulsion from palm oil esters was developed as a delivery system to produce nanocosmeceuticals. The stability of the resulting formulation was tested using various methods. In addition, the effect of components i.e. Vitamin E and Pluronic F-68 on the formulation was also studied.
  16. Structure and dynamics of Candida rugosa lipase: the role of organic solvent
    Tejo BA, Salleh AB, Pleiss J
    J Mol Model, 2004 Dec;10(5-6):358-66.
    PMID: 15597204
    The effect of organic solvent on the structure and dynamics of proteins was investigated by multiple molecular dynamics simulations (1 ns each) of Candida rugosa lipase in water and in carbon tetrachloride. The choice of solvent had only a minor structural effect. For both solvents the open and the closed conformation of the lipase were near to their experimental X-ray structures (C(alpha) rms deviation 1-1.3 A). However, the solvents had a highly specific effect on the flexibility of solvent-exposed side chains: polar side chains were more flexible in water, but less flexible in organic solvent. In contrast, hydrophobic residues were more flexible in organic solvent, but less flexible in water. As a major effect solvent changed the dynamics of the lid, a mobile element involved in activation of the lipase, which fluctuated as a rigid body about its average position. While in water the deviations were about 1.6 A, organic solvent reduced flexibility to 0.9 A. This increase rigidity was caused by two salt bridges (Lys85-Asp284, Lys75-Asp79) and a stable hydrogen bond (Lys75-Asn 292) in organic solvent. Thus, organic solvents stabilize the lid but render the side chains in the hydrophobic substrate-binding site more mobile. [figure: see text]. Superimposition of open (black, PDB entry 1CRL) and closed (gray, PDB entry 1TRH) conformers of C. rugosa lipase. The mobile lid is indicated.
  17. Fulltext Structure to function prediction of hypothetical protein KPN_00953 (Ycbk) from Klebsiella pneumoniae MGH 78578 highlights possible role in cell wall metabolism
    Teh BA, Choi SB, Musa N, Ling FL, Cun ST, Salleh AB, et al.
    BMC Struct Biol, 2014;14:7.
    PMID: 24499172 DOI: 10.1186/1472-6807-14-7
    Klebsiella pneumoniae plays a major role in causing nosocomial infection in immunocompromised patients. Medical inflictions by the pathogen can range from respiratory and urinary tract infections, septicemia and primarily, pneumonia. As more K. pneumoniae strains are becoming highly resistant to various antibiotics, treatment of this bacterium has been rendered more difficult. This situation, as a consequence, poses a threat to public health. Hence, identification of possible novel drug targets against this opportunistic pathogen need to be undertaken. In the complete genome sequence of K. pneumoniae MGH 78578, approximately one-fourth of the genome encodes for hypothetical proteins (HPs). Due to their low homology and relatedness to other known proteins, HPs may serve as potential, new drug targets.
  18. Danger lurking in the "unknowns": structure-to-function studies of hypothetical protein Bleg1_2437 from Bacillus lehensis G1 alkaliphile revealed an evolutionary divergent B3 metallo-beta-lactamase
    Tan SH, Normi YM, Leow AT, Salleh AB, Murad AM, Mahadi NM, et al.
    J. Biochem., 2017 02 01;161(2):167-186.
    PMID: 28175318 DOI: 10.1093/jb/mvw058
    The effectiveness of β-lactam antibiotics as chemotherapeutic agents to treat bacterial infections is gradually threatened with the emergence of antibiotic resistance mechanism among pathogenic bacteria through the production metallo-β-lactamase (MBL). In this study, we discovered a novel hypothetical protein (HP) termed Bleg1_2437 from the genome of alkaliphilic Bacillus lehensis G1 which exhibited MBL-like properties of B3 subclass; but evolutionary divergent from other circulating B3 MBLs. Domain and sequence analysis of HP Bleg1_2437 revealed that it contains highly conserved Zn2+-binding residues such as H54, H56, D58, H59, H131 and H191, important for catalysis, similar with the subclass B3 of MBL. Built 3-D Bleg1_2437 structure exhibited an αββα sandwich layer similar to the well-conserved global topology of MBL superfamily. Other features include a ceiling and floor in the model which are important for accommodation and orientation of β-lactam antibiotics docked to the protein model showed interactions at varying degrees with residues in the binding pocket of Bleg1_2437. Hydrolysis activity towards several β-lactam antibiotics was proven through an in vitro assay using purified recombinant Bleg1_2437 protein. These findings highlight the presence of a clinically important and evolutionary divergent antibiotics-degrading enzyme within the pools of uncharacterized HPs.
  19. Fulltext A Sco protein among the hypothetical proteins of Bacillus lehensis G1: Its 3D macromolecular structure and association with Cytochrome C Oxidase
    Tan SH, Normi YM, Leow AT, Salleh AB, Karjiban RA, Murad AM, et al.
    BMC Struct Biol, 2014 Mar 19;14:11.
    PMID: 24641837 DOI: 10.1186/1472-6807-14-11
    BACKGROUND: At least a quarter of any complete genome encodes for hypothetical proteins (HPs) which are largely non-similar to other known, well-characterized proteins. Predicting and solving their structures and functions is imperative to aid understanding of any given organism as a complete biological system. The present study highlights the primary effort to classify and cluster 1202 HPs of Bacillus lehensis G1 alkaliphile to serve as a platform to mine and select specific HP(s) to be studied further in greater detail.

    RESULTS: All HPs of B. lehensis G1 were grouped according to their predicted functions based on the presence of functional domains in their sequences. From the metal-binding group of HPs of the cluster, an HP termed Bleg1_2507 was discovered to contain a thioredoxin (Trx) domain and highly-conserved metal-binding ligands represented by Cys69, Cys73 and His159, similar to all prokaryotic and eukaryotic Sco proteins. The built 3D structure of Bleg1_2507 showed that it shared the βαβαββ core structure of Trx-like proteins as well as three flanking β-sheets, a 310 -helix at the N-terminus and a hairpin structure unique to Sco proteins. Docking simulations provided an interesting view of Bleg1_2507 in association with its putative cytochrome c oxidase subunit II (COXII) redox partner, Bleg1_2337, where the latter can be seen to hold its partner in an embrace, facilitated by hydrophobic and ionic interactions between the proteins. Although Bleg1_2507 shares relatively low sequence identity (47%) to BsSco, interestingly, the predicted metal-binding residues of Bleg1_2507 i.e. Cys-69, Cys-73 and His-159 were located at flexible active loops similar to other Sco proteins across biological taxa. This highlights structural conservation of Sco despite their various functions in prokaryotes and eukaryotes.

    CONCLUSIONS: We propose that HP Bleg1_2507 is a Sco protein which is able to interact with COXII, its redox partner and therefore, may possess metallochaperone and redox functions similar to other documented bacterial Sco proteins. It is hoped that this scientific effort will help to spur the search for other physiologically relevant proteins among the so-called "orphan" proteins of any given organism.

  20. A novel organic solvent tolerant lipase from Bacillus sphaericus 205y: extracellular expression of a novel OST-lipase gene
    Sulong MR, Abdul Rahman RN, Salleh AB, Basri M
    Protein Expr. Purif., 2006 Oct;49(2):190-5.
    PMID: 16769222
    An organic solvent tolerant (OST) lipase gene from Bacillus sphaericus 205y was successfully expressed extracellularly. The expressed lipase was purified using two steps purification; ultrafiltration and hydrophobic interaction chromatography (HIC) to 8-fold purity and 32% recovery. The purified 205y lipase revealed homogeneity on denaturing gel electrophoresis and the molecular mass was at approximately 30 kDa. The optimum pH for the purified 205y lipase was 7.0-8.0 and its stability showed a broad range of pH value between pH 5.0 to 13.0 at 37 degrees C. The purified 205y lipase exhibited an optimum temperature of 55 degrees C. The activity of the purified lipase was stimulated in the presence of Ca2+ and Mg2+. Ethylenediaminetetraacetic acid (EDTA) has no effect on its activity; however inhibition was observed with phenylmethane sulfonoyl fluoride (PMSF) a serine hydrolase inhibitor. Organic solvents such as dimethylsulfoxide (DMSO), methanol, p-xylene and n-decane enhanced the activity. Studies on the effect of oil showed that the lipase was most active in the presence of tricaprin (C10). The lipase exhibited 1,3 positional specificity.
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