Discovery And Engineering Of Fe(Ⅱ)/α-ketoglutarate-dependent Amino Acid Halogenase

The halogenated amino acids are important building blocks for synthesis of many functional chemicals including pharmaceuticals and agricultural chemicals.Fe（Ⅱ）/α-ketoglutarate（αKG）-dependent halogenases are the key enzymes for halogenation of non-aromatic amino acids,however only few halogenases have been reported,and they suffer from several problems such as low activities and narrow substrate spectra.By enzyme mining and enzymes engineering,we expect to discover new Fe（Ⅱ）/αKG dependent halogenases and improve their catalytic properties.Since Fe（Ⅱ）/αKG dependent amino acid hydroxylases and amino acid halogenases belong to the family of Fe（Ⅱ）/αKG dependent oxygenases,new amino acid halogenases may be obtained by engineering such amino acid hydroxylases.Thus,the Fe（Ⅱ）/αKG dependent trans-proline-4-hydroxylase Da P4H was engineered in this study,and five point mutants were designed and cloned.Among them,the Da P4H-D111G and Da P4H-F209L mutants showed activities towards L-proline,and the structures of products remain to be further elucidated.To discover new Fe（Ⅱ）/αKG dependent amino acid halogenases,six putative amino acid halogenase genes were predicted and synthesized after gene mining and sequence similarity network analysis in this study.The plasmids harboring the putative halogenase gene were transformed into Escherichia coli for heterologous enzyme expression and purification.After the transformation of amino acid catalyzed by the purified enzymes,the high performance liquid chromatography（HPLC）analysis showed that Ca Hal,Ak Hal and AtHal had obvious activities towards L-lysine（activity order:Ak Hal>AtHal>Ca Hal）.Considering the relatively higher thermostability and novelty（sequence identity with Bes D:53%）,the AtHal was selected for further study.The engineering work can be divided into two parts:three engineering strategies（the sequence conservation analysis,similar amino acid scanning and loop replacement）were employed to improve the activity of AtHal towards L-lysine;the loop replacement and point mutations were employed to expand the substrate spectra of AtHal.Consequently,the kinetic parameters of the purified enzymes towards L-lysine showed that four AtHal mutants had higher catalytic efficiency than wild-type,and k_cat/K_Mof AtHal-HA（34.50±1.00）was 4.8-fold higher than wild-type（7.09±0.61）,k_cat/K_Mof H160A（16.90±0.92）was 2.4-fold higher than wild-type,k_cat/K_Mof T247S（10.03±0.77）was 1.4-fold higher than wild-type,and k_cat/K_Mof AtHal-HA/T19（9.36±1.31）was 1.3-fold higher than wild-type.On the other hand,the activities of AtHal mutants towards L-leucine and L-ornithine were determined using enzyme-coupled method.As a result,AtHal-LEU showed activity towards L-leucine,and mutants e6,e5 and g5 had activities（order:e6>e5>g5）towards L-ornithine,however all their activities were low.In summary,this study identified the key residues and regions affecting the catalytic properties of Fe（Ⅱ）/αKG dependent amino acid halogenases,which may guide the further engineering of these enzymes.