Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (3): 75-83.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0830

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Functional Analysis of Key Residues in the Active Center of Creatinase from Alcaligenes sp. KS-85

HAO Jun-yao1(), MA Fu-qiang1,2, YANG Guang-yu1   

  1. 1. State Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai JiaoTong University,Shanghai 200240
    2. CAS Key Lab of Bio-Medical Diagnostics,Suzhou Institute of Biomedical Engineering and Technology,Chinese Academy of Sciences,Suzhou 215163
  • Received:2020-07-07 Online:2021-03-26 Published:2021-04-02

Abstract:

Creatinase(CRE)hydrolyses creatine to urea and sarcosine,and plays an important role in the enzymatic measurement of creatinine concentration for in vitro diagnosis. To better understand the catalytic mechanism of CRE,the homology modeling,molecular docking and alanine scanning were used to analyze the interaction between enzyme and substrate. Focusing on 4 function-unknown key residues Phe64,Asp102,Phe252,and Phe321 in the active center of the enzyme,site-directed mutagenesis was conducted to mutate them into 6 representative amino acids,and their functions were analyzed by combing the bio-chemical experiment. The kcat values of all mutants significantly decreased compared with the wild type enzyme. The KM of F252A,F252S,F252Y,F252W,F321A,and F321Y decreased while that of others increased. Structural analysis showed that Phe252 stabilized the enzyme-substrate complex by the π-π stacking with Tyr259. Asp102 stabilized the transition state of the enzyme reaction by interacting with the hydrogen bonds of Arg66 and Gly322. Phe321 and Phe64 were located at two sides of the substrate,which affected substrate positioning by their hydrophobicity and steric hindrance. This study lays a foundation for understanding the catalytic mechanism and molecular engineering of creatinase via the functional analysis of amino acid residues in the active center.

Key words: creatinase, active center, catalytic efficiency, molecular docking, functional residues