Study On The Mechanism Of The Key Sites Of Alanine Metabolism Related Enzymes

D-alanine is an important chiral intermediate,widely used in food,cosmetics,physiology,biopharmaceutical and so on.Alanine dehydrogenase and Alanine racemase are the key enzyme proteins in the synthesis and metabolism of D-alanine in peptidoglycan of bacterial cell wall,also as a D-alanine biosynthetic component in industrial production.In this study,Alanine dehydrogenase(OF4Ald)and Alanine racemase(MBalr,OF4dadX)from Bacillus pseudolfirmus OF4 and Thermoanaerobacter tengcongensis MB4 were studied,on the base of three-dimensional analysis in structure,structural simulation,site-directed mutagenesis and characterization of enzymatic properties,explore the mechanism of the key site of enzyme proteins and the catalytic mechanism,provide a theoretical basis for the reform and obtain D-alanine biosynthesis components with high activity and high stability.It was found that alanine dehydrogenase from B.pseudofirmus OF4 may have five highly conserved active sites R15,K73,K75,H96 and D269 by homologous sequence alignment,the mutant R15A,K75A,H96A and D269A completely lost their activity through site-directed mutagenesis and detection of enzyme activity.It was presumed that these four sites could be the active sites of alanine dehydrogenase.However,the relative activity of K73A was about twice that of wild type,the 73 site was subtituted to K73E,K73F,K73Q,K73S,K73R and K73Y,respectively.By measuring the kinetic parameters of the mutant and combining with the homologous structure simulation technique,it was found that the K73 site interacted with the K75 site through the hydrogen bond and affected the binding of the enzyme to the substrate.When the hydrogen bond was present,the affinity of the enzyme proteins and substrate was weak,when the hydrogen bond was disappear,the affinity was strong.According to the literature,there are eight highly conserved amino acid sites in the substrate entryway of alanine racemase.However,there are only six conservative sites in the biosynthetic alanine racemase from T.tengcongensis MB4,and the remaining two sites are nonconservative amino acids.In order to explore the mechanism of the nonconservative sites,the sites were replaced by conservative amino acid residues:S173D and Q360Y,the results of mutant activity showed that the catalytic efficiency of the mutant Q360Y was 17.83 times that of the wild type,which indicated that the Q360 site might be the key site of the catalytic activity of the enzyme proteins.Although the catalytic efficiency of S173D was only 80%of the wild type,the optimal temperature of the mutant S173D was 80?,which was 15? higher than that of the wild type.The S173 site was further studied by semi-saturated mutation.The optimal temperature of the 173 mutant was higher than that of the wild type about 5-15?,the result indicated that the S173 site was the key site of enzyme proteins,which affected optimal temperature of the racemase.The interaction between the conservative site A172 and S173,Q360 in the MBalr substrate entryway was also investigated.On the base of the mutants S173D,Q360H and Q360Y,2-point mutants and 3-point mutants were constructed by site-directed mutagenesis.The results showed that A172 was replaced with S172,which resulted in the relative activity of mutant enzyme proteins had been improved(3.16%-11.92%),but the stability of these mutants was poor,Q360 site could improve the activity of enzyme protein,its stability was also different with different amino acids to replaced,S173 site could enhance the stability by reducing the catalytic activity of enzyme.In short,Q360 site may be involved in regulating the activity of alanine racemase,5173 site may be involved in regulation of the stability,while the S173 and Q360 sites are associated with the selectivity of the substrateBased on the results of three-dimensional structure analysis of alanine racemase OF4dadX,it was found that there were five amino acid sites D43,D70,E71,E134 and D318 in the dimer interface of alanine racemase(OF4dadX),which participated in the formation of the salt bonds.In order to explore the mechanism of these five amino acids,the five sites were replaced with Lysine(K)by site-directed mutagenesis to destroy the salt bonds,the relative activity and the low aggregative state of the enzyme proteins were determined.The results showed that D43K and D70K were similar to the wild-type about the relative activity and the low aggregative state(dimer),while E71K,E134K and D318K were lost activity and the relative molecular weight were about 45.7-48.9 kDa,which were close to monomer molecular weight of OF4dadX.It is suggested that the salt bonds was formed by E71,E134 and D318,that may be involved in OF4dadX dimerization,maintain the dimer structure and spatial configuration.After the mutation,the enzyme protein was in the form of monomer,and the catalytic activity of the enzyme protein was lost.