Structure And Catalytic Mechanism Studies Of Acetolactate Decarboxylase From Bacillus Subtilis

Acetolactate decarboxylase(ALDC)catalyzes(R)-and(S)-enantiomers of acetolactateto generate a single product,(R)-acetoin.The rapid development of green chemistry has made more and more obvious advantages of microbial fermentation in the chemical industry,especially the rapid progress in the application of non-pathogenic microorganisms in food,medicine and other fields.Although the biosynthesis of acetoin has been achieved through the use of synthetic biology and metabolic engineering strategies,the stereoselective molecular mechanism of the substrate and product which remains unclear.As yet rare atomic level structures of ALDC are present despite the enzyme is widely existing in microorganisms,except the ever-reported X-ray crystal structure of ALDC from Bacillus brevis and Staphylococcus aureus.In this paper,ALDC from Bacillus subtilis(B.s.-ALDC)transformed into E.coli BL21.ALDC proteins were induced by IPTG with a soluble expression at 37 oC.Through ion exchange chromatography and gel filtration chromatography,the ALDC with high purity was obtained.The purified B.s.-ALDC was characterized by matrix-assisted laser desorption /ionization(MALDI),experimental molecular mass of B.s.-ALDC is 28983.4 Da.The calculated specific activity of B.s.-ALDC was 115.16 U/mg.Measured the activity of B.s.-ALDC using a colorimetric method,Voges-Proskauer(VP)assay.The sitting drop vapor diffusion method was adapted to screen B.s.-ALDC crystal grow conditions,the final diffraction data was collected from a crystal grown in 0.1 M sodium chloride,0.1 M Tris,p H 8.5,25%(w/v)PEG 3350 at a protein concentration of 30 mg/m L.We solved and reported a 1.5 ? resolution crystal structure of B.s.-ALDC.Initial indexing of the diffraction patterns indicated that B.s.-ALDC crystallized in the P31 space group with unit cell dimensions: a=71.303 ?,b=71.303 ? and c=84.13 ?(? = ? = 90° and ? = 120°),Rfree=0.202,Rwork=0.200.Two molecules are present per asymmetric unit,dimeric assembly is observed in the solved structure.A zinc ion is coordinated by highly conserved histidines(191,193 and 204),together with conserved glutamates(62 and 251).We used both enantiomers of acetolactate as substrates to further investigate the substrate bias of B.s.-ALDC by means of molecular docking and dynamic simulation in silico.The binding free energy of(S)-?-acetolactate is ~30 kcal/mol greater than that of(R)-?-acetolactate,indicating a more stable binding for(S)-?-acetolactate.In conclusion,the recombinant expression of ALDC from Bacillus subtilis 168 was achieved,the crystal structure was obtained,the structural information of B.s.-ALDC was revealed at the atomic level,and the key amino acid residues of determining the stereoselectivity in the catalytic process were determined.A mechanism for the transformation of each enantiomer of acetolactate is analyzed preliminarily.