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  1. Enhancement in T1 lipase purification recovery using the novel construct pGEX4T1/His-T1
    Hussian CHAC, Rahman RNZRA, Leow ATC, Salleh AB, Ali MSM, Latip W
    Prep Biochem Biotechnol, 2024 Apr;54(4):526-534.
    PMID: 37647127 DOI: 10.1080/10826068.2023.2252052
    The Geobacillus zalihae strain T1 produces a thermostable T1 lipase that could be used for industrial purposes. Previously, the GST-T1 lipase was purified through two chromatographic steps: affinity and ion exchange (IEX) but the recovery yield was only 33%. To improve the recovery yield to over 80%, the GST tag from the pGEX system was replaced with a poly-histidine at the N-terminal of the T1 lipase sequence. The novel construct of pGEX/His-T1 lipase was developed by site-directed mutagenesis, where the XbaI restriction site was introduced upstream of the GST tag, allowing the removal of tag via double digestion using XbaI and EcoRI (existing cutting site in the pGEX system). Fragment of 6 × His-T1 lipase fusion was synthesized, cloned into the pGEX4T1 system, and expressed in Escherichia coli BL21 (DE3) pLysS, resulting in lipase-specific activity at 236 U/mg. The single purification step of His-T1 lipase was successfully achieved using nickel Sepharose 6FF with an optimized concentration of 5 mM imidazole for binding, yielding the recovery of 98%, 1,353 U/mg lipase activity, and a 5.7-fold increase in purification fold. His-T1 lipase was characterized and was found to be stable at pH 5-9, active at 70 °C, and optimal at pH 9.
    Matched MeSH terms: Mutagenesis, Site-Directed
  2. Molecular cloning, homology modeling and site-directed mutagenesis of vanadium-dependent bromoperoxidase (GcVBPO1) from Gracilaria changii (Rhodophyta)
    Baharum H, Chu WC, Teo SS, Ng KY, Rahim RA, Ho CL
    Phytochemistry, 2013 Aug;92:49-59.
    PMID: 23684235 DOI: 10.1016/j.phytochem.2013.04.014
    Vanadium-dependent haloperoxidases belong to a class of vanadium enzymes that may have potential industrial and pharmaceutical applications due to their high stability. In this study, the 5'-flanking genomic sequence and complete reading frame encoding vanadium-dependent bromoperoxidase (GcVBPO1) was cloned from the red seaweed, Fracilaria changii, and the recombinant protein was biochemically characterized. The deduced amino acid sequence of GcVBPO1 is 1818 nucleotides in length, sharing 49% identity with the vanadium-dependent bromoperoxidases from Corralina officinalis and Cor. pilulifera, respectively. The amino acid residues associated with the binding site of vanadate cofactor were found to be conserved. The Km value of recombinant GcVBPO1 for Br(-) was 4.69 mM, while its Vmax was 10.61 μkat mg(-1) at pH 7. Substitution of Arg(379) with His(379) in the recombinant protein caused a lower affinity for Br(-), while substitution of Arg(379) with Phe(379) not only increased its affinity for Br(-) but also enabled the mutant enzyme to oxidize Cl(-). The mutant Arg(379)Phe was also found to have a lower affinity for I(-), as compared to the wild-type GcVBPO1 and mutant Arg(379)His. In addition, the Arg(379)Phe mutant has a slightly higher affinity for H2O2 compared to the wild-type GcVBPO1. Multiple cis-acting regulatory elements associated with light response, hormone signaling, and meristem expression were detected at the 5'-flanking genomic sequence of GcVBPO1. The transcript abundance of GcVBPO1 was relatively higher in seaweed samples treated with 50 parts per thousand (ppt) artificial seawater (ASW) compared to those treated in 10 and 30 ppt ASW, in support of its role in the abiotic stress response of seaweed.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  3. 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.
    Matched MeSH terms: Mutagenesis, Site-Directed
  4. Fulltext Bacillus thuringiensis subsp. Entomocidus HD9 Cry1Ba4 insecticidal crystal protein: in-silico mutation, cloning, expression, mutation and domain I functional study
    Mohd Fadzli Ahmad, Hasdianty Abdullah
    Bioremediation Science and Technology Research, 2015;3(2):20-25.
    MyJurnal
    The 3D structure of the insecticidal protein Cry1Ba4 produced by B. thuringiensis subsp.
    Entomocidus HD-9 was determined using homology modelling. From the model built, we have
    been able to identify the possible sites for structure modification by site-directed mutagenesis.
    The mutation was introduced at the conserved region of -helix 7 by substituting the
    hydrophobic motif that comprises alanine 216, leucine 217 and phenylalanine 218 with arginine.
    Wild and mutant Cry1Ba4 genes were cloned into pET200/D-TOPO and expressed in the
    expression host. The result suggests that mutant Cry1Ba4 protein was less toxic to the larvae
    Plutella xylostella compared to the wild-type. In conclusion, alteration in the structure of
    Domain I had left an impact on the toxicity of Cry1Ba4 against P. xylostella.
    Matched MeSH terms: Mutagenesis, Site-Directed
  5. Site-directed mutagenesis of β sesquiphellandrene synthase enhances enzyme promiscuity
    Ker DS, Chan KG, Othman R, Hassan M, Ng CL
    Phytochemistry, 2020 May;173:112286.
    PMID: 32059132 DOI: 10.1016/j.phytochem.2020.112286
    The chemical formation of terpenes in nature is carried out by terpene synthases as the main biocatalysts to guide the carbocation intermediate to form structurally diverse compounds including acyclic, mono- and multiple cyclic products. Despite intensive study of the enzyme active site, the mechanism of specific terpene biosynthesis remains unclear. Here we demonstrate that a single mutation of the amino acid L454G or L454A in the active site of Persicaria minor β-sesquiphellandrene synthase leads to a more promiscuous enzyme that is capable of producing additional hydroxylated sesquiterpenes such as sesquicineole, sesquisabinene hydrate and α-bisabolol. Furthermore, the same L454 residue mutation (L454G or L454A) in the active site also improves the protein homogeneity compared to the wild type protein. Taken together, our results demonstrate that residue Leucine 454 in the active site of β-sesquiphellandrene synthase is important for sesquiterpene product diversity as well as the protein homogeneity in solution.
    Matched MeSH terms: Mutagenesis, Site-Directed
  6. Fulltext Development of a pyrG mutant of Aspergillus oryzae strain S1 as a host for the production of heterologous proteins
    Ling SO, Storms R, Zheng Y, Rodzi MR, Mahadi NM, Illias RM, et al.
    ScientificWorldJournal, 2013;2013:634317.
    PMID: 24381522 DOI: 10.1155/2013/634317
    The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5'-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. β -Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous β -galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.
    Matched MeSH terms: Mutagenesis, Site-Directed/methods*
  7. Fulltext Rational mutagenesis of cyclodextrin glucanotransferase at the calcium binding regions for enhancement of thermostability
    Goh PH, Illias RM, Goh KM
    Int J Mol Sci, 2012;13(5):5307-23.
    PMID: 22754298 DOI: 10.3390/ijms13055307
    Studies related to the engineering of calcium binding sites of CGTase are limited. The calcium binding regions that are known for thermostability function were subjected to site-directed mutagenesis in this study. The starting gene-protein is a variant of CGTase Bacillus sp. G1, reported earlier and denoted as "parent CGTase" herein. Four CGTase variants (S182G, S182E, N132R and N28R) were constructed. The two variants with a mutation at residue 182, located adjacent to the Ca-I site and the active site cleft, possessed an enhanced thermostability characteristic. The activity half-life of variant S182G at 60 °C was increased to 94 min, while the parent CGTase was only 22 min. This improvement may be attributed to the formation of a shorter α-helix and the alleviation of unfavorable steric strains by glycine at the corresponding region. For the variant S182E, an extra ionic interaction at the A/B domain interface increased the half-life to 31 min, yet it reduced CGTase activity. The introduction of an ionic interaction at the Ca-I site via the mutation N132R disrupted CGTase catalytic activity. Conversely, the variant N28R, which has an additional ionic interaction at the Ca-II site, displayed increased cyclization activity. However, thermostability was not affected.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  8. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis
    Hussain H, Chong NF
    Biomed Res Int, 2016;2016:8041532.
    PMID: 27995143
    The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40-45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment.
    Matched MeSH terms: Mutagenesis, Site-Directed/methods*
  9. Site-directed mutagenesis: role of lid region for T1 lipase specificity
    Mohamed RA, Salleh AB, Leow TC, Yahaya NM, Abdul Rahman MB
    Protein Eng. Des. Sel., 2018 06 01;31(6):221-229.
    PMID: 30239965 DOI: 10.1093/protein/gzy023
    A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region. BSLA lipase was chosen as the reference protein for T1 lipase modification since it contains no lid. Initially, structures of both enzymes were compared via protein-protein superimposition in 3D-space and the location of the lid region of T1 lipase was highlighted. A total of three variants of T1 lipase without lid were successfully designed by referring to BSLA lipase (a lipase without lid). The ability of T1 lipase without lid variants in degrading P(3HB) was investigated quantitatively. All the variants showed activity towards the substrate which confirmed that T1 lipase without lid is indeed able to degrade P(3HB). In addition, D2 was recorded to have the highest activity amongst other variants. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation but also P(3HB) by simply removing the lid region.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  10. Fulltext Homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerantPseudomonassp. AMS8
    Garba L, Mohamad Yussoff MA, Abd Halim KB, Ishak SNH, Mohamad Ali MS, Oslan SN, et al.
    PeerJ, 2018;6:e4347.
    PMID: 29576935 DOI: 10.7717/peerj.4347
    Membrane-bound fatty acid desaturases perform oxygenated desaturation reactions to insert double bonds within fatty acyl chains in regioselective and stereoselective manners. The Δ9-fatty acid desaturase strictly creates the first double bond between C9 and 10 positions of most saturated substrates. As the three-dimensional structures of the bacterial membrane fatty acid desaturases are not available, relevant information about the enzymes are derived from their amino acid sequences, site-directed mutagenesis and domain swapping in similar membrane-bound desaturases. The cold-tolerantPseudomonassp. AMS8 was found to produce high amount of monounsaturated fatty acids at low temperature. Subsequently, an active Δ9-fatty acid desaturase was isolated and functionally expressed inEscherichia coli. In this paper we report homology modeling and docking studies of a Δ9-fatty acid desaturase from a Cold-tolerantPseudomonassp. AMS8 for the first time to the best of our knowledge. Three dimensional structure of the enzyme was built using MODELLER version 9.18 using a suitable template. The protein model contained the three conserved-histidine residues typical for all membrane-bound desaturase catalytic activity. The structure was subjected to energy minimization and checked for correctness using Ramachandran plots and ERRAT, which showed a good quality model of 91.6 and 65.0%, respectively. The protein model was used to preform MD simulation and docking of palmitic acid using CHARMM36 force field in GROMACS Version 5 and Autodock tool Version 4.2, respectively. The docking simulation with the lowest binding energy, -6.8 kcal/mol had a number of residues in close contact with the docked palmitic acid namely, Ile26, Tyr95, Val179, Gly180, Pro64, Glu203, His34, His206, His71, Arg182, Thr85, Lys98 and His177. Interestingly, among the binding residues are His34, His71 and His206 from the first, second, and third conserved histidine motif, respectively, which constitute the active site of the enzyme. The results obtained are in compliance with thein vivoactivity of the Δ9-fatty acid desaturase on the membrane phospholipids.
    Matched MeSH terms: Mutagenesis, Site-Directed
  11. Fulltext Enhancement of a protocol purifying T1 lipase through molecular approach
    Che Hussian CHA, Raja Abd Rahman RNZ, Thean Chor AL, Salleh AB, Mohamad Ali MS
    PeerJ, 2018;6:e5833.
    PMID: 30479887 DOI: 10.7717/peerj.5833
    T1 Lipase is a thermostable secretary protein of Geobacillus zalihae strain previously expressed in a prokaryotic system and purified using three-step purification: affinity 1, affinity 2, and ion exchange chromatography (IEX). This approach is time consuming and offers low purity and recovery yield. In order to enhance the purification strategy of T1 lipase, affinity 2 was removed so that after affinity 1, the cleaved Glutathione S-transferase (GST) and matured T1 lipase could be directly separated through IEX. Therefore, a rational design of GST isoelectric point (pI) was implemented by prediction using ExPASy software in order to enhance the differences of pI values between GST and matured T1 lipase. Site-directed mutagenesis at two locations flanking the downstream region of GST sequences (H215R and G213R) was successfully performed. Double point mutations changed the charge on GST from 6.10 to 6.53. The purified lipase from the new construct GST tag mutant-T1 was successfully purified using two steps of purification with 6,849 U/mg of lipase specific activity, 33% yield, and a 44-fold increase in purification. Hence, the increment of the pI values in the GST tag fusion T1 lipase resulted in a successful direct separation through IEX and lead to successful purification.
    Matched MeSH terms: Mutagenesis, Site-Directed
  12. 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.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  13. Fulltext Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli
    Jonet MA, Mahadi NM, Murad AM, Rabu A, Bakar FD, Rahim RA, et al.
    J. Mol. Microbiol. Biotechnol., 2012;22(1):48-58.
    PMID: 22456489 DOI: 10.1159/000336524
    A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  14. 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.
    Matched MeSH terms: Mutagenesis, Site-Directed/methods*
  15. Site-saturation mutagenesis of mutant L-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase
    Ismail A, Illias RM
    J Ind Microbiol Biotechnol, 2017 Dec;44(12):1627-1641.
    PMID: 28921081 DOI: 10.1007/s10295-017-1980-6
    The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  16. Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function
    Goh KM, Liew KJ, Chai KP, Illias RM
    Methods Mol Biol, 2017;1498:385-396.
    PMID: 27709591
    Protein engineering is a very useful tool for probing structure-function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.
    Matched MeSH terms: Mutagenesis, Site-Directed/methods
  17. Characterization of Chlamydomonas Ribulose-1,5-bisphosphate carboxylase/oxygenase variants mutated at residues that are post-translationally modified
    Rasineni GK, Loh PC, Lim BH
    Biochim Biophys Acta Gen Subj, 2017 Feb;1861(2):79-85.
    PMID: 27816753 DOI: 10.1016/j.bbagen.2016.10.027
    BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the chloroplast enzyme that fixes CO2 in photosynthesis, but the enzyme also fixes O2, which leads to the wasteful photorespiratory pathway. If we better understand the structure-function relationship of the enzyme, we might be able to engineer improvements. When the crystal structure of Chlamydomonas Rubisco was solved, four new posttranslational modifications were observed which are not present in other species. The modifications were 4-hydroxylation of the conserved Pro-104 and 151 residues, and S-methylation of the variable Cys-256 and 369 residues, which are Phe-256 and Val-369 in land plants. Because the modifications were only observed in Chlamydomonas Rubisco, they might account for the differences in kinetic properties between the algal and plant enzymes.

    METHODS: Site-directed mutagenesis and chloroplast transformation have been used to test the essentiality of these modifications by replacing each of the residues with alanine (Ala). Biochemical analyses were done to determine the specificity factors and kinetic constants.

    RESULTS: Replacing the modified-residues in Chlamydomonas Rubisco affected the enzyme's catalytic activity. Substituting hydroxy-Pro-104 and methyl-Cys-256 with alanine influenced Rubisco catalysis.

    CONCLUSION: This is the first study on these posttranslationally-modified residues in Rubisco by genetic engineering. As these forms of modifications/regulation are not available in plants, the modified residues could be a means to modulate Rubisco activity.

    GENERAL SIGNIFICANCE: With a better understanding of Rubisco structure-function, we can define targets for improving the enzyme.

    Matched MeSH terms: Mutagenesis, Site-Directed/methods
  18. Site-Directed Mutagenesis of Cytochrome P450 2D6 and 2C19 Enzymes: Expression and Spectral Characterization of Naturally Occurring Allelic Variants
    Dong AN, Pan Y, Palanisamy UD, Yiap BC, Ahemad N, Ong CE
    Appl Biochem Biotechnol, 2018 Sep;186(1):132-144.
    PMID: 29524040 DOI: 10.1007/s12010-018-2728-0
    Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
    Matched MeSH terms: Mutagenesis, Site-Directed*
  19. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture
    Steuernagel B, Periyannan SK, Hernández-Pinzón I, Witek K, Rouse MN, Yu G, et al.
    Nat Biotechnol, 2016 Jun;34(6):652-5.
    PMID: 27111722 DOI: 10.1038/nbt.3543
    Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5-15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.
    Matched MeSH terms: Mutagenesis, Site-Directed/methods*
  20. Fulltext Enzymatic properties and mutational studies of chalcone synthase from Physcomitrella patens
    Rahman RN, Zakaria II, Salleh AB, Basri M
    Int J Mol Sci, 2012;13(8):9673-91.
    PMID: 22949824 DOI: 10.3390/ijms13089673
    PpCHS is a member of the type III polyketide synthase family and catalyses the synthesis of the flavonoid precursor naringenin chalcone from p-coumaroyl-CoA. Recent research reports the production of pyrone derivatives using either hexanoyl-CoA or butyryl-CoA as starter molecule. The Cys-His-Asn catalytic triad found in other plant chalcone synthase predicted polypeptides is conserved in PpCHS. Site directed mutagenesis involving these amino acids residing in the active-site cavity revealed that the cavity volume of the active-site plays a significant role in the selection of starter molecules as well as product formation. Substitutions of Cys 170 with Arg and Ser amino acids decreased the ability of the PpCHS to utilize hexanoyl-CoA as a starter molecule, which directly effected the production of pyrone derivatives (products). These substitutions are believed to have a restricted number of elongations of the growing polypeptide chain due to the smaller cavity volume of the mutant's active site.
    Matched MeSH terms: Mutagenesis, Site-Directed
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