title: Design of a zinc-finger hydrolase with a synthetic αββ protein.
abstract: Recent advances in protein design have opened avenues for the creation of artificial enzymes needed for biotechnological and pharmaceutical applications. However, designing efficient enzymes remains an unrealized ambition, as the design must incorporate a catalytic apparatus specific for the desired reaction. Here we present a de novo design approach to evolve a minimal carbonic anhydrase mimic. We followed a step-by-step design of first folding the main chain followed by sequence variation for substrate binding and catalysis. To optimize the fold, we designed an αββ protein based on a Zn-finger. We then inverse-designed the sequences to provide stability to the fold along with flexibility of linker regions to optimize Zn binding and substrate hydrolysis. The resultant peptides were synthesized and assessed for Zn and substrate binding affinity by fluorescence and ITC followed by evaluation of catalytic efficiency with UV-based enzyme kinetic assays. We were successful in mimicking carbonic anhydrase activity in a peptide of twenty two residues, using p-nitrophenyl acetate as a CO2 surrogate. Although our design had modest activity, being a simple structure is an advantage for further improvement in efficiency. Our approach opens a way forward to evolving an efficient biocatalyst for any industrial reaction of interest.
authors.affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai, India.
authors.forename: Kinshuk Raj
authors.initials: KR
authors.lastname: Srivastava
authors.affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Mumbai, India.
authors.forename: Susheel
authors.initials: S
authors.lastname: Durani
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D032701:Biomimetics / Q000379:methods; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D006867:Hydrolases / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D066244:Proton Magnetic Resonance Spectroscopy; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010455:Peptides; C008644:nitrophenylphosphate; C008642:4-nitrophenyl acetate; D006867:Hydrolases; D002256:Carbonic Anhydrases; D015032:Zinc