Exchanging the active site between phytases for altering the functional properties of the enzyme

Lehmann, Martin; Lopez-Ulibarri, Rual; Loch, Claudia; Viarouge, Céline; Wyss, Markus; Loon, A. P. G. M. Van

doi:10.1110/ps.9.10.1866

Cited by 32 publications

(14 citation statements)

References 30 publications

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“…4C). A similar shift in pH optimum with the K68A mutation and with phytic acid as a substrate was also seen for the A. fumigatus Q27I variant and for several consensus phytases (consensus phytase-1 Q27T, consensus phytase-1 thermo [8] Q27T, and consensus phytase-10 thermo[3] Q27T; Fig. 4B and data not shown; for a detailed description of the different consensus phytases, see references 7 and 9 and M. Lehmann, C. Loch, A. Middendorf, D. Studer, S. F. Lassen, L. Pasamontes, A. P. G. M. van Loon, and M. Wyss, unpublished data).…”

Section: Resultssupporting

confidence: 63%

“…In addition, we have determined the crystal structure of A. niger phytase at a 2.5-Å resolution (4). Inspection of this three-dimensional (3D) structure, comparison with other high-M r histidine acid phosphatases, and site-directed mutagenesis of residues in the presumed active site (8,25,25) yielded strong evidence for a highly positively charged cleft at the interface between the smaller ␣-domain and the larger ␣/␤-domain of the molecule as the substrate binding (active) site.…”

Section: Resultsmentioning

confidence: 99%

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Engineering of Phytase for Improved Activity at Low pH

Tomschy

Brugger

Lehmann

et al. 2002

Appl Environ Microbiol

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For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.Active sites typically contain ionizable groups (Arg, Lys, His, Glu, and Asp) that are involved in substrate or product binding and/or catalysis and that determine the pH activity profile of an enzyme (2). Both for physiological constraints and for industrial applications, it is crucial that an enzyme works properly at the appropriate pH value(s). However, no truly reliable methods for modifying the pH activity profile of an enzyme are yet available. In order to understand more clearly how nature masters catalysis at physiological pH values and to rationally tailor enzymes for industrial use at a predefined pH value, it is important to gain more experimental experience on the pH optimum engineering of enzymes.Different strategies can be chosen to modify the pH activity profile of an enzyme. (i) The first is the replacement of ionizable groups that are directly involved in substrate or product binding and/or catalysis by nonionizable ones or by amino acids with different charge or pK values (i.e., "A residues" [13,23]).(ii) The second is the replacement of residues that are in direct contact with A residues by forming hydrogen bonds and/or salt bridges. Substitution of such residues may disturb the hydrogen-bonding network in the active site or alter the electronic environment of A residues (1,11,19,27). The effects on the pH activity profile caused by this type of mutations are particularly difficult to predict. (iii) The third is the alteration of...

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Section: Resultssupporting

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Engineering of Phytase for Improved Activity at Low pH

Tomschy

Brugger

Lehmann

et al. 2002

Appl Environ Microbiol

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“…Chen et al also discussed the role of noncovalent intramolecular interactions in the thermostability of barnase [19]. Some experimental studies have reported that phytase thermostability was enhanced using various methods, including the introduction of a consensus sequence [20,21], rational design [16,22], directed evolution [23,24], and the introduction of hydrogen bonds or ionic interactions [13,17]. Using MD simulation, numerous sites and regions in many proteins have been recognized as enhancing thermal stability [17,[25][26][27][28][29][30][31][32].…”

Section: Introductionmentioning

confidence: 99%

Insights into the unfolding pathway and identification of thermally sensitive regions of phytase from Aspergillus niger by molecular dynamics simulations

Kumar¹,

Patel

Agrawal

et al. 2015

J Mol Model

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Thermal stability is of great importance in the application of commercial phytases. Phytase A (PhyA) is a monomeric protein comprising twelve α-helices and ten β-sheets. Comparative molecular dynamics (MD) simulations (at 310, 350, 400, and 500 K) revealed that the thermal stability of PhyA from Aspergillus niger (A. niger) is associated with its conformational rigidity. The most thermally sensitive regions were identified as loops 8 (residues 83-106), 10 (161-174), 14 (224-230), 17 (306-331), and 24 (442-444), which are present on the surface of the protein. It was observed that solvent-exposed loops denature before or show higher flexibility than buried residues. We observed that PhyA begins to unfold at loops 8 and 14, which further extends to loop 24 at the C-terminus. The intense movement of loop 8 causes the helix H2 and beta-sheet B3 to fluctuate at high temperature. The high flexibility of the H2, H10, and H12 helices at high temperature resulted in complete denaturation. The high mobility of loop 14 easily transfers to the adjacent helices H7, H8, and H9, which fluctuate and partially unfold at high temperature (500 K). It was also observed that the salt bridges Asp110-Lys149, Asp205-Lys277, Asp335-Arg136, Asp416-Arg420, and Glu387-Arg400 are important influences on the structural stability but not the thermostability, as the lengths of these salt bridges did not increase with rising temperature. The salt bridges Glu125-Arg163, Asp299-Arg136, Asp266-Arg219, Asp339-Lys278, Asp335-Arg136, and Asp424-Arg428 are all important for thermostability, as the lengths of these bridges increased dramatically with increasing temperature. Here, for the first time, we have computationally identified the thermolabile regions of PhyA, and this information could be used to engineer novel thermostable phytases. Numerous homologous phytases of fungal as well as bacterial origin are known, and these homologs show high sequence similarity. Our findings could prove useful in attempts to increase the thermostability of homologous phytases via protein engineering.

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“…1). Consensus-7 phytase (Lehmann et al, 2000b), produced by substitution of the active site of Anp for its counterpart in consensus-1 phytase (Lehmann et al, 2000a), exhibits a major, although not complete, shift in pH-activity profile towards Anp, and a much greater T m (70.4 • C). Despite the same active site shared by Anp, chimera ABCDEFG, and consensus-7 phytase, Anp possesses the highest level of specific activity.…”

Section: Discussionmentioning

confidence: 99%

“…Many positions influencing the catalytic properties of Anp or Afp have been identified by site-directed mutagenesis on the basis of 3D structure and sequence alignment (Wyss et al, 1999b;Tomschy et al, 2000aTomschy et al, ,b, 2002Mullaney et al, 2002;Kim et al, 2006;Zhang and Lei, 2008). All these positions are located within the theoretical catalytic pocket (Lehmann et al, 2000b). Among them, however, only positions E205, K277 and H282 (all positions are numbered according to the amino acid numbering of mature Anp (Kostrewa et al, 1997) on the basis of sequence alignment) are responsible for the differences in substrate specificity and activity in the acidic pH range between Anp and Afp (Tomschy et al, 2002;Mullaney et al, 2002;Kim et al, 2006).…”

Section: Introductionmentioning

confidence: 99%