Cloning And Functional Analysis Of The Gene Encoding Novel 3,6-dihydroxypicolinic Acid Decarboxylase In Alcaligenes Faecalis JQ135

Picolinic acid(PA),a typical C2-carboxylated pyridine derivative,is a metabolite of L-tryptophan and many other aromatic compounds in mammalian and microbial cells.Some high-efficiency pyridine herbicides in agricultural production,such as picloram and diquat,are easily decomposed,yielding large amounts of PA.Due to its stability and long half-life in the environment,PA has caused a potential harm to ecosystems and human health.Previous studies have shown that microbial metabolism is the main contribution of PA degradation in the environment.Many microorganisms can degrade and grow with PA as the sole carbon,nitrogen and energy source.However,the specific microbial metabolic mechanism of PA has not been elucidated.Several studies only speculated that PA could be partially degraded by adding hydroxyl groups to form 6-hydroxypicolinic acid(6HPA),which is then decarboxylated to 2,5-dihydroxypyridine(2,5DHPA).However,it is speculated that the intermediate product of 3,6-dihydroxypicolinic acid(3,6DHPA)may exist between 6HPA and 2,5DHP according to the law of substance metabolism.In addition,there are no reports on the degradation of related enzymes and genes.Therefore,it is of great theoretical and practical value to elucidate the microbial degradation and metabolism mechanism of PA from the level of genes and enzymology.In this study,the PA-degradation strain Alcaligenes faecalis JQ135 and its mutant strain Mut-H4 were used as the research materials.The inactivated genes were cloned by SEFA-PCR and compared with the whole genome of wild strain JQ135,a possible 3,6DHPA degradation gene picC was found.Through the knockout and complement of the gene,purification of heterologous expression,identification of the metabolites and the studies of enzymatic properties,we revealed the microbial catabolism of an important intermediate 3,6DHPA during the PA degradation.The related coding genes and enzymes were also involved.The discoveries of this study further improved the microbial metabolic pathway of PA,and opened up the in-depth exploration of microbial degradation from the level of genes and enzymology.The main results of this paper are mainly divided into the following aspects:1.Degradation characteristics of the mutant strain Mut-H4In the previous study,we screened a 6HPA degradation-deficient mutant strain Mut-H4 through random transposon mutagenesis.The strain was found to be able to metabolize PA to form 6HPA by adding hydroxyl groups,and eventually accumulated 6HPA into an intermediate.The intermediate was identified as 3,6DHPA by UV-VIS,HPLC and LC-MS/MS analysis.It was speculated that the mutant strain Mut-H4 lost the genes related to the metabolism of 3,6DHPA.2.Cloning of 3,6DHPA degradation geneThe inactivated gene was cloned by SEFA-PCR and compared with the whole genome of wild strain A.faecalis JQ135.We found that the gene picC was responsible for catalyzing the degradation of 3,6DHPA.The full length of the gene is 972 bp,encoding 323 amino acids.The amino acid sequence analysis of PicC showed that the enzyme belongs to the amidohydrolase₂ family.picC exhibited the highest sequence similarity to y-resorcylate decarboxylase(?-RSD,45%identity)from Rhizobium sp.strain MTP-10005.The similarity is low,so PicC forms a separate branch in the phylogenetic tree.3.Function identification of 3,6DHPA decarboxylase PicCThe knockout strain pJQ-?picC deficient in 3,6DHPA degradation activity,was successfully constructed by the gene knockout method.Then the picC gene was cloned into the wide host vector pBBR1MCS5 to obtain the complement plasmid pBBR-picC,which was then transformed into the knockout strain pJQ-?picC to regain the ability to degrade 3,6DHPA.These results further confirmed that picC is the gene responsible for encoding 3,6DHPA decarboxylase.In addition,the knockout and complement of gene also proved that picC is essential for the degradation of PA.4.Enzymatic properties of 3,6DHPA decarboxylase PicCThe picC gene was expressed heterologously in E.coli and the protein was purified.The identification of 3,6DHPA metabolized by PicC indicated that the enzyme could catalyze the decarboxylation of 3,6DHPA and convert it to 2,5DHP.The Km,Vmax and kcat values of 3,6DHPA catalyzed by PicC were 13.44 ?M,7.73?mol·min-1·mg-1 and 4.77 s-1,respectively.The catalytic efficiency kcat/Km was 0.35 ?M-1·s-1.The optimum reaction conditions of 3,6DHPA decarboxylase PicC were 40?,pH 7.0.Some metal ions could inhibit the activity of PicC in varying degrees.Hg2+,Ag+ and Cu2+ could strongly inhibit the activity of PicC(>70%),while the inhibition of Ca2+,Co2+,Mg2+,Mn2+,Zn2+,Fe3+ and Cd2+ were weak.Further,ICP-OES analysis revealed the presence of Zn2+ at 0.85±0.1 mol per mol of protein,which was similar to several non-oxidative decarboxylases of the amidohydrolase₂ superfamily.These results indicated the presence of Zn2+ in the center of PicC,PicC had high specificity for substrate 3,6DHPA and no catalytic activity for structural analogues.The above studies indicated that PicC was a Zn2+-dependent non-oxidative decarboxylase that can specifically catalyze the decarboxylation of 3,6DHPA to 2,5DHP.5.Studies on Key Amino Acid Sites of 3,6DHPA Decarboxylase PicCBy adding some common amino acid inhibitors to detect their effects on the catalytic activity of PicC.It was found that DEPC,a histidine residue modifier,could strongly inhibit the decarboxylation activity of PicC.It was speculated that the histidine in PicC played a key role in the process of catalytic decarboxylation of 3,6DHPA.Further comparison of the amino acid sequences of PicC and its seven homologous decarboxylases,revealed that these proteins have seven conserved histidine sites.The seven conserved histidines(His)in PicC were mutated to alanine(Ala)by overlap extension PCR,and these mutant genes were heterologously expressed and purified.The catalytic activity of these mutant proteins to 3,6DHPA were also determined.It was found that the decarboxylase activity of PicCH135A was slightly higher than PicC,while the activity of PicCH172A and PicCH194A was slightly lower(about 10-50%).The activities of PicCH12A and PicCH177A decreased significantly(about 90%)while the mutant proteins PicCH163A and PicCH216A lost their catalytic activities for decarboxylation of 3,6DHPA completely.These results showed that His 163 and His216 were critical for the activity of PicC.