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Chappell, Todd C.

Krishna, Naveen Banchallihundi

Kumar, Pravin

Mohan, Karishma

Nair, Nikhil U.

Ramesh, Athreya

Shetty, Anuj

Sigamani, Gladstone G.

Trivedi, Vikas D.

English

Engineering Conferences International

 Poster Number 21 QM/MM STUDIES OF THE PHENYLALANINE AMMONIA-LYASE VARIANTS HELPED TO UNDERSTAND THE MECHANISTIC ROLE OF THE MUTATIONS  Naveen Banchallihundi Krishna, Kcat Enzymatic Private Limited, Bangalore, India naveen.bk@kcat.co.in Anuj Shetty, Kcat Enzymatic Private Limited, Bangalore, India Pravin Kumar R, Kcat Enzymatic Private Limited, Bangalore, India Gladstone G. Sigamani, Kcat Enzymatic Private Limited, Bangalore, India Vikas D. Trivedi, Department of Chemical and Biological Engineering, Tufts University, USA Todd C. Chappell, Department of Chemical and Biological Engineering, Tufts University, , USA  Karishma Mohan, Department of Chemical and Biological Engineering, Tufts University, USA  Athreya Ramesh, Department of Chemical and Biological Engineering, Tufts University, , USA Nikhil U. Nair Department of Chemical and Biological Engineering, Tufts University, USA   Key words: PAL, QM/MM, Metadynamics  Pegvaliase, the only FDA-approved drug for treating classical phenylketonuria (PKU), is a PEGylated Phenylalanine ammonia-lyase (PAL). PAL from Anabaena variabilis, which known for the conversion of l-phenylalanine to trans-cinnamic acid and has widespread application in chemoenzymatic synthesis, agriculture, and medicine. Deep mutational scanning (DMS) has recently emerged as a powerful method to study protein sequence–function relationships which used for identifying hotspots and mutations to improve reaction kinetics in cell-free and cellular contexts. Behavioural studies of enzyme after the incorporation of the mutations were thoroughly studied using quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) studies.  QM/MM studies were conducted on 4 chains of the enzyme simultaneously which helped to understand the mechanistic role of the mutations including stabilizing transition and intermediate states. Molecular Dynamics Based Metadynamics studies with unique collective Variable (CV) helped us to understand changes in substrate binding affinity among mutations and umbrella sampling was used to understand the association and dissociation path of the substrate which helped to reduce product inhibition.      References: 1. In-Depth Sequence–Function Characterization Reveals Multiple Pathways to Enhance Enzymatic Activity Vikas D. Trivedi, Todd C. Chappell, Naveen B. Krishna, Anuj Shetty, Gladstone G. Sigamani, Karishma Mohan, Athreya Ramesh, Pravin Kumar R, and Nikhil U. Nair. ACS Catalysis 2022 12 (4), 2381-2396, DOI: 10.1021/acscatal.1c05508   Figure 1. QM/MM implemented in this study (A) 1 μs MCV equilibrated E-S conformation was extended with QMMM-PM7 simulation using NAMD 2.13 to derive the attack conformation. This reduced the distance between Y-OH and NH3-Sub from ~3.5 Å to 1.5 Å in all the 4 chains. (B) Post this, TS level TS optimizations were adapted to obtain transition state and the final state  

QM/MM Studies of The phenylalanine ammonia-lyase variants helped to understand the mechanistic role of the mutations

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QM/MM Studies of The phenylalanine ammonia-lyase variants helped to understand the mechanistic role of the mutations

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Engineering Conferences International