Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions

AbstractBackground: Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes.Results: The crystal structure of substrate-free LigAB was solved at 2.2 Å resolution. The molecule is an α2β2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the β subunit, which is covered by the α subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site.Conclusions: The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism

A bacterial enzyme degrading the model lignin compound β-etherase is a member of the glutathione-S-transferase superfamily

AbstractCleavage of β-aryl ether linkages is essential in lignin degradation. We identified another β-etherase gene (ligF), which contains an open reading frame of 771 bp and lies between genes coding Cα-dehydrogenase (ligD) and β-etherase (ligE). The β-etherase activity of LigF expressed in Escherichia coli was more than 80 times as high as that of LigE. ligF and ligE are homologous to glutathione-S-transferase, and upon addition of glutathione a remarkable acceleration of β-etherase activity was found in E. coli carrying ligF. It is concluded that LigF plays a central role in β-aryl ether cleavage and that glutathione is the hydrogen donor in this reaction

Discovery of novel enzyme genes involved in the conversion of an arylglycerol-β-aryl ether metabolite and their use in generating a metabolic pathway for lignin valorization

Microbial conversions known as “biological funneling” have attracted attention for their ability to upgrade heterogeneous mixtures of low-molecular-weight aromatic compounds obtained by chemical lignin depolymerization. β-hydroxypropiovanillone (HPV) and its analogs can be obtained by chemoselective catalytic oxidation of lignin using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O2, followed by cleavage of arylglycerol-β-aryl ether with zinc. Sphingobium sp. strain SYK-6 can degrade HPV generated by the catabolism of arylglycerol-β-aryl ether through 2-pyrone-4,6-dicarboxylate (PDC), a promising platform chemical. Therefore, production of PDC from HPV can be achieved using the HPV catabolic pathway. However, the pathway and genes involved in the catabolism of vanilloyl acetic acid (VAA) generated during HPV catabolism have not been investigated. In the present study, we isolated SLG_24960 (vceA), which encodes an enzyme that converts VAA into a coenzyme A (CoA) derivative of vanillate (vanilloyl-CoA) from SYK-6, by shotgun cloning. The analysis of a vceA mutant indicated that this gene is not required for VAA conversion in vivo, but it encodes a major enzyme catalyzing CoA-dependent VAA conversion in vitro. We also identified SLG_12450 (vceB), whose product can convert vanilloyl-CoA to vanillate. Enzyme genes besides vceA and vceB, which are necessary for the conversions of HPV to VAA and of vanillate to PDC, were introduced and expressed in Pseudomonas putida. The resulting engineered strain completely converted 1 mM HPV into PDC after 24 h. Our results suggest that the enzyme genes that are not required for the catabolic pathway in microorganisms but can be used for the conversion of target substrates are buried in microbial genomes. These genes are, thus, useful for designing metabolic pathways to produce value-added metabolites.PostprintPeer reviewe

Multiple-Subunit Genes of the Aromatic-Ring-Hydroxylating Dioxygenase Play an Active Role in Biphenyl and Polychlorinated Biphenyl Degradation in Rhodococcus sp. Strain RHA1

A gram-positive strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, can degrade PCBs by cometabolism with biphenyl or ethylbenzene. In RHA1, three sets of aromatic-ring-hydroxylating dioxygenase genes are induced by biphenyl. The large and small subunits of their terminal dioxygenase components are encoded by bphA1 and bphA2, etbA1 and etbA2, and ebdA1 and ebdA2, respectively, and the deduced amino acid sequences of etbA1 and etbA2 are identical to those of ebdA1 and ebdA2, respectively. In this study, we examined the involvement of the respective subunit genes in biphenyl/PCB degradation by RHA1. Reverse transcription-PCR and two-dimensional polyacrylamide gel electrophoresis analyses indicated the induction of RNA and protein products of etbA1 and ebdA1 by biphenyl. Single- and double-disruption mutants of etbA1, ebdA1, and bphA1 were constructed by insertional inactivation. The 4-chlorobiphenyl (4-CB) degradation activities of all the mutants were lower than that of RHA1. The results indicated that all of these genes are involved in biphenyl/PCB degradation. Furthermore, we constructed disruption mutants of ebdA3 and bphA3, encoding ferredoxin, and etbA4, encoding ferredoxin reductase components. The 4-CB degradation activities of these mutants were also lower than that of RHA1, suggesting that all of these genes play a role in biphenyl/PCB degradation. The substrate preferences of etbA1A2/ebdA1A2- and bphA1A2-encoded dioxygenases for PCB congeners were examined using the corresponding mutants. The results indicated that these dioxygenase isozymes have different substrate preferences and that the etbA1A2/ebdA1A2-encoded isozyme is more active on highly chlorinated congeners than the bphA1A2-encoded one

Transcriptional Regulation of the Terephthalate Catabolism Operon in Comamonas sp. Strain E6 ▿

Two almost identical gene clusters, tphRICIA2IA3IBIA1I and tphRIICIIA2IIA3IIBIIA1II, are responsible for the conversion of terephthalate (TPA) to protocatechuate in Comamonas sp. strain E6. In the present study, we investigated the transcriptional regulation of the tphRIICIIA2IIA3IIBIIA1II gene cluster. Reverse transcription-PCR analysis suggested that the tphRIICIIA2IIA3IIBIIA1II genes form two transcriptional units, the tphCIIA2IIA3IIBIIA1II catabolism operon and tphRII, with the latter encoding an IclR-type transcriptional regulator (ITTR). The transcription start site of the tphII catabolism operon was mapped at 21 nucleotides upstream of the initiation codon of tphCII. The lacZ transcriptional fusion experiments showed that tphRII encodes a transcriptional activator of the tphII catabolism operon and that TPA acts as an inducer. On the other hand, TphRII appeared to repress its own transcription regardless of the presence of TPA. The analysis of mutant derivatives of E6 indicated that tphRII is essential for the transcriptional activation of the tphII catabolism operon and the growth on TPA of a tphI-deficient derivative of E6. Purified His-tagged TphRII bound specifically to the tphRII-tphCII intergenic region containing a 21-bp inverted repeat sequence. Alignment of the inverted repeat sequences in the binding sites for TphRII and other members of ITTRs revealed highly conserved nucleotides. The substitution of conserved nucleotides resulted in significantly reduced TPA-dependent transcriptional activation from the tphCII promoter and reduced binding to His-tagged TphRII. These results clearly indicate that the conserved nucleotides are required for the inducible expression of the tphII catabolism operon regulated by TphRII