Molecular Mechanism Of The Catalytic Inactivation Of L-aspartate Alpha-decarboxylase

Amino acid decarboxylase is a family of lyases that catalyzes the decarboxylation of amino acids to form small molecular amino acids or biogenic amines which mostly have special physiological functions in organism.Among them,L-aspartate alpha-decarboxylase?EC:4.1.1.11,PanD/ADC?serves as one of the most popular decarboxylases,as it could catalyze the alpha-decarboxylation of L-aspartic acid to generate beta-alanine and CO₂.Beta-alanine,as an important platform compound,is a precursor for the synthesis of several bulk chemicals such as calcitonin pantothenate,1,4-butanediamine,acrylamide,and so on,which has been widely used in the fields of pharmaceutical,food and chemical engineering and has great industrial value.Currently,compared with industrial chemical conversion routs,the green manufacture routs of beta-alanine by PanD and cheap raw material L-aspartic acid has gradually become the mainstream technology.However,the gene sources of industrially-used PanD limited in Escherichia coli,Corynebacterium glutamate or Bacillus subtilis,and all of these PanDs showed irreversible inactivation during the catalytic process which was called catalytic inactivation.However,the mechanism of irreversible inactivation cannot be clearly explained as yet.This problem resulted in low production efficiency and hindered the development of related biological industry.In this paper,a large number of PanD from various sources were acquired by constructing a hypothetic protein library of PanD.By kinetic analysis,the mechanism of catalytic deactivation of PanD was analyzed,and the characterization method of catalytic deactivation intensity was established.Then,the structural basis of catalytic inactivation was identified depending on two aspects:self-cleavage process and catalytic domain.The main research results are as follows:?1?By constructing a hypothetic protein library,53 PanDs ecoding genes from different sources were screened.By searching and phylogenetic analysis of L-aspartate alpha-decarboxylase encoding genes in GenBank biological information database,a hypothetic protein library of PanD was established,and 38 of PanD were successfully expressed in Escherichia coli.After purified and charactered in vitro,33 of them were identified to show the PanD activity.Besides,by summarizing the expression,protein post-translation process,and catalytic inactivation of PanD in the library,it was indicated that all pyruvoyl-dependent L-aspartate alpha-decarboxylase?PYR-ADC?showed significant catalytic inactivation,while pyridoxal phosphate-dependent L-aspartate alpha-decarboxylase?PLP-ADC?did not.In addition,8PanDs were screened by determination of specific activity from the library.Among them,PanD from Corynebacterium jeikeium(PD_Cjei)showed the highest specific activity?11.55U/mg?reported as yet.?2?Based on kinetic analysis results,the irreversibility of catalytic deactivation in PYR-ADC was found,and the determination method on the intensity of catalytic inactivation was established.By monitoring the catalytic process of PanD and the effect of substrate concentration on the bioconversion of PanD from different sources in the library,and also the residual activity determined after ultrafiltration,the inrreversible inactivation of PanD by L-aspartate was verified.Besides,time-and L-aspartate concerntration-dependent inactivation was observed which indicatied that the inactivation of PanD was a mechanism-based inactivation reaction.The value K_I and k_inactnact were determined as 288.4 mM and 0.163 min^-1,respectively.The logarithm of residual activity within 500 mM of substrate concentration was linearly correlated with reaction time,and the slope of the Napierian logarithm value of the residual activity vs.time was the observed inactivation rate(k_obs)which could be used to characterize the intensity of substrate inactivation.?3?Key residues on the catalytic inactivation-dependent self-cleavage mechanism of PYR-ADC were identified based on the library.PanDs from 33 sources with purity of no less than 90%were purified by HisTrap HP chromatography and analyzed by Tricine-SDS-PAGE.Spectrophotometric scanning of electrophoretogram was performed using Image Lab^TM software,and the self-cleavage efficiency of PanD from different sources was compared based on the percentage of alpha-and beta-subunit bands.All PanDs in the library were divided into three classes based on the self-cleavage efficiency.Combined with phylogenetic analysis results,distinct sequence divergence was found around the cleavage site:the amino acid sequence of EGSCA was conserved in Class I,while that of VGSIT was conserved in Class III.Further study on point mutation and structure comprarison represented that four conserved residues?Val23,Ile26,Thr27 and Glu56?play an important role in self-cleavage of PanD.?4?Key residues on the catalytic inactivation-dependent functional domain were identified based on the library.Mutation of 12 residues were selected and analyzed according to the discrepency in catalytic stability and protein structure of PanD in the library.By point saturation mutation and colorimetric high throughput sceening method,four mutants with improved specific activity were accquied.Further kinetic analysis indicated that Arg3,Ala74,and Glu42 were the key residues on catalytic activity,and Arg3 and Ala74 also played an important role on the catalytic inactivation of PD_Cjei.Besides,compared with that of wild-type,R3K showed significant improvement with its catalytic efficiency increased by 3 times and catalytic stability increased by 66.38%.?5?Characterization and application of non-catalytic inactivated PanD selected from the library.Pyridoxal phosphate-dependent PanD?PLP-ADC?ecoding genes?6 items?were found in the database and expressed in E.coli.After purificatoin and characterization,3 PLP-ADCs from the archaea show appliable PanD activity.The specific activity of PLP-ADC from Methanocaldococcus jannaschii(PD_Mja)was 6.87 U/mg which was the highest among PLP-ADCs.Enzymatic properties indicated that PD_Mja showed great thermal stability;the optimum temperature and the optimum pH were 80oC and 8.0,respectively.Bioconversion results showed that PLP was degraded by the substrate during catalyzation,which makes the stability and recycling of the coenzyme PLP the main limiting factors for the application of PDMja.