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Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution

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Abstract

Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE,  an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The “best” combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure–function relationships of enzymes.

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Acknowledgments

We thank BASF SE for financial support.

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Authors and Affiliations

  1. Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    Amol V. Shivange & Ulrich Schwaneberg

  2. School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany

    Amol V. Shivange, Danilo Roccatano & Ulrich Schwaneberg

  3. Present Address: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, USA

    Amol V. Shivange

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  1. Amol V. Shivange
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  2. Danilo Roccatano
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Correspondence to Ulrich Schwaneberg.

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ESM 1

Fig. S1. PROCHECK-Ramachandran plot for the homology model of Ymphytase. Fig. S2 3D-profile window plots for Ymphytase homology model. The vertical axis shows the average 3D-1D score for the residue and horizontal line indicate residue number in the model. The residues with average positive score in 3D-1D slide window are reasonably folded. Fig. S3 Radius of gyration of the backbone atoms (Cα, N, C) of Ymphytase throughout the simulation of wild-type (a; Wt) and variant M6 (b). Fig. S4 Structural topology of Ymphytase model obtained from molecular dynamics simulation. Fig. S5 Hydrogen bonding network of Ymphytase wild-type deciphered by MD simulation. Amino acid residue G187 showed no hydrogen bonds with loop-G6 (a), T77 did not show any hydrogen bonds with D122 (b), K289 showed 2 – 3 hydrogen bonds with helix-L (c) but the frequency of second hydrogen bond was less compare to variant M6 and negligible hydrogen bonds with loop-CD (d). Fig. S6 Salt bridge analysis of T77K with D122 showing the minimum distance between residues K77 and D122 throughout the simulation time. The average distance between K77 and D122 was 0.37 nm showing a salt bridge interaction (centroids of the side-chain charged group atoms in the residues lie within 0.4 nm of each other). Fig. S7 A tetrameric model of Ymphytase constructed by superimposing monomers to biological assembly of D. castellii phytase (PDB ID: 2GFI). Substitutions in Ymphytase variant M6 are shown in orange colored space filled model. Highly flexible loops observed in MD simulation (L92-T102, N247-P252, and Q304-Q317) are shown in red color. Residues involved in inter-monomer interaction calculated using PIC server (http://crick.mbu.iisc.ernet.in/∼PIC/) are depicted in purple (hydrophobic interactions), yellow (ionic interactions), and blue (hydrogen bonding interactions) color. Fig. S8 Hydrogen bonding network of Ymphytase residue Q154 in wild-type (a) and substitution Q154H in variant M6 over the time of MD simulation. Hydrogen bonds were calculated between residue 154 and the protein molecule. Table S1 Sequence of the primers used for site-directed and site saturation mutagenesis. Table S2 Classification and mutational pattern of the variants identified in the 2nd round of SeSaM library screening. Table S3 Available templates for homology modeling of YmPhytase (*selected template). Fig. S9 Structure-sequence alignment of template and query sequence based on dynamic programming algorithm of MODELLER for YmPhytase. Fig. S10 Activity-pH profile of the Ymphytase wildtype and variant M3 and M6. (DOCX 2227 kb)

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Shivange, A.V., Roccatano, D. & Schwaneberg, U. Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution. Appl Microbiol Biotechnol 100, 227–242 (2016). https://doi.org/10.1007/s00253-015-6959-5

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