Unravelling the gallic acid degradation pathway in bacteria: the gal cluster from Pseudomonas putida

Abstract

SummaryGallic acid (3,4,5-trihydroxybenzoic acid, GA) is widely distributed in nature, being a major phenolic pollutant and a commonly used antioxidant and building-block for drug development. We have characterized the first complete cluster (gal genes) responsible for growth in GA in a derivative of the model bacterium Pseudomonas putida KT2440. GalT mediates specific GA uptake and chemotaxis, and highlights the critical role of GA transport in bacterial adaptation to GA consumption. The proposed GA degradation via the central intermediate 4--oxalomesaconic acid (OMA) was revisited and all enzymes involved have been identified. Thus, GalD is the prototype of a new subfamily of isomerases that catalyses a biochemical step that remained unknown, i.e. the tautomerization of the OMAketo generated by the GalA dioxygenase to OMAenol. GalB is the founding member of a new family of zinc-containing hydratases that converts OMAenol into 4-carboxy-4-hydroxy-2-oxoadipic acid (CHA). galC encodes the aldolase catalysing CHA cleavage to pyruvic and oxaloacetic acids. The presence of homologous gal clusters outside the Pseudomonas genus sheds light on the evolution and ecology of the gal genes in GA degraders. The gal genes were used for expanding the metabolic abilities of heterologous hosts towards GA degradation, and for engineering a GA cellular biosensor.

Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440

“…The molecular mass of the GalA, GalB, GalD, and LigJ CsE6 subunits, as determined by SDS-PAGE, was ϳ45, ϳ25, ϳ40, and ϳ40 kDa (data not shown), which is consistent with the predicted molecular mass values of 47.6, 27.5, 37.6, and 38.2 kDa, respectively, from previous reports (9,10,19,20).…”

“…DNA Manipulations-pET29a plasmids harboring galA, galB, and galD from P. putida KT2440 were a generous gift from E. Díaz, Biological Research Center-CSIC, Madrid, Spain (9). GalB variants A16D, R21A, E48A, R67A, H127A, Y123A, H164A, and R216A were produced by site-specific mutagenesis using the QuikChange method (16).…”

“…Assays for CHM hydration and CHA dehydration were completed similarly to that described previously (9). Briefly, the hydration of CHM to CHA, or the dehydration of CHA to CHM, was monitored by detecting the olefinic signal of CHM at 265 nm.…”

“…In contrast, galD homologs are frequently found in lig operons and is found in the same gene cluster as the canonical protocatechuate 4,5-cleavage pathway of Sphingo- bium sp. SYK-6 (9,47). The presence of a GalD homolog in the Lig pathways suggests that the product of 2-pyrone-4,6-dicarboxylate (PDC) lactonase, LigI, is OMA rather than (2Z,4E)-CHM, which is subsequently tautomerized to (2E,4E)-CHM by the GalD homolog to be used by either GalB or LigJ (Fig.…”

“…Like LigJ, GalB has no sequence similarity to the previously characterized divalent metal-dependent hydratases. The initial characterization of the gallate pathway identified (2E,4E)-CHM as the substrate for GalB, whereas (2Z,4E)-CHM is the proposed substrate for LigJ (8,9). The proposed differences in substrate specificity between the two enzymes would give a biological rationale for the two possibly distinct CHM hydratases.…”