Para-hydroxybenzoate Catabolic Pathway With A Key Step Involving An Intramolecular Migration Of Carboxyl Group In Brevibacillus Laterosporus PHB-7a

The NIH shift is a chemical rearrangement in which a substituent on an aromatic ring undergoes an intramolecular migration,primarily during an enzymatic hydroxylation reaction catalyzed by enzyme（s）.This phenomenon is found to be involved in disorder of amino acid metabolism,metabolism of therapeutic drugs,antibiotics synthesis and degradation of aromatic compounds.The substituents subject to NIH shifts include hydrogen,halogen,aceto group,alkyl group and carboxyl group.The 1,2-shift of hydrogen and aceto groups have been well studied and the molecular mechanisms are established,in which single oxygenase was involved for both cases.But the mechanisms of other groups such as carboxyl group still remains unresolved.The molecular mechanism for the NIH shift of a carboxyl group has remained a mystery for forty years.The intramolecular migration of carboxyl group is found in the aerobic catabolism of para-hydroxybenzoate（PHB）.PHB is a natural compound widely released into the environment mainly through lignin degradation and has also been established to be involved in the full pathogenicity of plant pathogenic bacteria.PHB can also be efficiently utilized as carbon source for growth by many microorganisms.The aerobic utilization of PHB has been studied well,the mainly aerobic catabolic pathways include protocatechuate pathway,hydroquinone pathway and gentisate pathway（GA）which involving an NIH shift of carboxyl group.The molecular mechanisms of the first two pathways has been studied well,but the genetic and enzymatic reports for the NIH shift of a carboxyl group has not been documented.Brevibacillus laterosporus PHB-7a degrades PHB via GA.In this study,the genes and enzymes involved in the 1,2-shift of the carboxyl group during the transformation of PHB to GA in strain PHB-7a was established,and the intermediates produced by enzymes were also identified.We detailedly clarified a novel intramolecular migration mechanism and proposed a PHB-GA catabolic pathway,which is more complicated than previously thought.The genes and enzymes involved in the further catabolism of GA to the TCA cycle intermediates were also determined.Firstly,genome sequencing and bioinformatics analysis revealed a bagXILK cluster of four genes involved in the GA catabolism.These four genes were heterologously expressed and BagI,BagL,BagK were purified.Functional identification showed that BagX,BagI,BagL and BagK were meta-hydroxybenzoate（MHB）6-hydroxylase（1.34 U/mg against MHB）,gentisate 1,2-dioxygenase（5.54U/mg against gentisate）,L-cysteine-dependent maleylpyruvate isomerase（1.98 U/mg against maleylpyruvate）and fumarylpyruvate hydrolase（2.98 U/mg against fumarylpyruvate）,respectively,indicating that the gentisate was catabolized to TCA cycle intermediates via an isomerization pathway in strain PHB-7a.Secondly,after the genes involved in PHB catabolism via GA in strain PHB-7a was determined to be inducibly expressed,transcriptome analyses of differential genes expression revealed a phgABC cluster involved in the conversion of PHB to GA.Heterologous expression experiments were conducted to establish rigorously that PhgABC has the ability to transform PHB to GA.When stable isotope labeled PHB(2,3,5,6-tetradeutero-PHB and[carboxyl-¹³C]-PHB)were used as the substrates for biotransformation,the analysis results of the stable isotope labeled GA established the migration and retention of the original carboxyl group,which migrated from the C1position to C2 position.This is the first time to have clearly established the carboxyl group migration at genetic levels.Finally,phgA,phgB,phgC were heterologously expressed and purified.After the chemical synthesis of p-hydroxybenzoyl-CoA,the function of PhgA,PhgB,PhgC were identified and the enzymatic kinetic parameters of PhgC were determined.Functional identification showed that PhgA is a p-hydroxybenzoyl-CoA hydroxylase,PhgB is a gentisyl-CoA thioesterase and PhgC is a p-hydroxybenzoyl-CoA ligase.These three enzymes catalyze the conversion of PHB to GA via thioester formation,hydroxylation and thioester hydrolysis.This is the first time to reveal the carboxyl group migration at enzymatic levels.The essence of carboxyl group migration is the 1,2 shift of acyl-CoA moiety of p-hydroxybenzoyl-CoA induced by the hydroxylation of aromatic ring.Bioinformatics analysis also revealed that the gene cluster of phgABC seems to be only found in Bacillus,and the phgABC cluster in strain PHB-7a may have obtained from Bacillus butanolivorans AFS003229 through the way of horizontal gene transfer.The NIH shift in carboxyl group migration achieved is distinct from those involved in migration of hydrogen and aceto groups,where a single oxygenase catalyzes the reaction without involvement of a thioester.The discovery of this three-step strategy for carboxyl group migration reveals a novel role of the thioester in biochemistry and also illustrates the diversity and complexity of microbial catabolism in the carbon cycle.