Characterization of EstZY: A new acetylesterase with 7-aminocephalosporanic acid deacetylase activity from Alicyclobacillus tengchongensis

Int J Biol Macromol. 2020 Apr 1:148:333-341. doi: 10.1016/j.ijbiomac.2020.01.151. Epub 2020 Jan 16.

Abstract

Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic β-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC2 to p-NPC8. Maximum enzyme activity was recorded at pH 9.0 and 50 °C, where Km and Vmax were calculated as 1.9 ± 0.23 mM and 258 ± 18.5 μM min-1, respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3' position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values.

Keywords: 7-Aminocephalosporanic acid; Acetylesterase; Antibiotics.

MeSH terms

  • Acetylesterase / metabolism*
  • Alicyclobacillus / metabolism*
  • Amino Acid Sequence
  • Cephalosporins / metabolism*
  • Cloning, Molecular / methods
  • Esterases / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Docking Simulation / methods
  • Sequence Alignment
  • Substrate Specificity

Substances

  • Cephalosporins
  • 7-aminocephalosporanic acid
  • Esterases
  • Acetylesterase

Supplementary concepts

  • Alicyclobacillus tengchongensis