Gene Mining And Biological Function Of Aldo-keto Reductase From Myxobacteria

Aldo-keto reductase(AKR)is a NAD(P)H-dependent redox enzyme,which is widely distributed in fungi,bacteria,animals and plants.AKR is widely used in chemical and pharmaceutical fields because of its broad substrate spectrum and asymmetric reduction of carbonyl compounds to form corresponding chiral alcohols.AKR is also related to the biological function of the organism,such as the occurrence of various cancers,glycometabolism and detoxification of aldehydes and ketones.Ethyl chloro-3-hydroxybutyrate(CHBE)is an important intermediate for the synthesis of drugs.Asymmetric catalysis of ethyl 4-chloroacetoacetate(COBE)by aldo-keto reductase is one of the effective ways to synthesize CHBE.In this paper,based on the genome sequence of myxobacteria Corallocnccus sp.EGB,the akr genes were screened.The akrs were cloned and solubly expressed.The recombinant enzyme CoAKR7 which can catalyze COBE was selected for further study.CoAKR7 was NADPH-dependent,the gene sequence was 882 bp in length,encoding 293 amino acids and with a predicted molecular weight of 31.33 kDa.The recombinant enzyme was purified by Ni affinity chromatography and its enzymatic properties were studied.The results showed that the optimal reaction temperature and pH of CoAKR7 was 50? and 7.0(Tris-HCl).The enzyme exhibited good stability of temperature and pH,and it retained 80%activity after being incubated at 40? for 60 min.I mM Fe2and Fe3+,5 mM Cu2+and Cr3+,10%of SDS had a strong inhibitory effect on enzyme activity.The enzyme displayed high stereoselectivity to COBE,producing S-CHBE with the e.e.value of 99%.The substrate specificity indicated that the CoAKR7 showed catalytic activity to substrates of ketoesters and aromatic ketones,with less than 20%activity to COBE.The determination of kinetic parameters showed that CoAKR7 had higher affinity for COBE,the Km and Vmax was 0.269 mM and 6.087 ?mol/min/mg,respectively.Molecular docking results showed that Arg202 and Asn257 played an important role in the preference of the enzyme for binding of NADPH.The chlorine atom of COBE forms a halogen-? bond with Tyr80,allowing the substrate to bind to CoAKR7 in a Prelog dominant conformation.Since the genetic manipulation system of strain EGB has not been established,strain DK1622 was initially explored for the potential function of akr genes in the physiological process of myxobacteria.The transcription of AKR genes at different development time were analyzed by RT-PCR with oar as control.4 akr genes were screened,and the corresponding mutants were constructed.Results showed that the growth and social motility of the four mutant strains did not change significantly.However,the developmental process of the mutant strain of MXAN_RS01910 gene was significantly delayed compared with strain DK1622 indicating that the gene plays a role in the fruiting body development of the myxobacteria.Further studies on this mutant showed that at 36 h and 60 h of development,the spore germination rate of the mutant strain was significantly lower than that of the wild strain.Lipid body staining at different developmental stages showed that,the content of lipid bodies in the mutant strain was significantly lower than the wild strain,and the speed of shortening of the mutant cell was also slower than that of the wild strain during development.At 40 h,the difference was the most significant,and the cell length of the defective strain was about twice that of DK1622.The lipids from strain DK1622 and mutants were extracted,and the same dose of extracts from both strains has been used to complement the mutant.The results showed that the effect of lipid extracts derived from strain DK1622 was slightly better than that of the mutant strain.It is presumed that the gene may be involved in the synthesis of lipids through the E signaling pathway in myxobacteria.