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Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDOKF1 from Comamonas testosteroni KF1 and found that it had an apparent kcat/Km for phthalate of 0.58 ± 0.09 μM−1s−1, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 A resolution revealed that it is a hexamer comprising two stacked α3 trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDOKF1 larger than that of other characterized ROs. Complexes of PDOKF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.

Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼106 M−1 s−1) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 A resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

Strain SYK-6 of the bacterium Sphingobium sp. catabolizes lignin-derived biphenyl via a meta-cleavage pathway. In this pathway, LigY is proposed to catalyze the hydrolysis of the meta-cleavage product (MCP) 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate. Here, we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2,4-dienoate as the products and determined the kcat and kcat/Km values as 9.3 ± 0.6 s-1 and 2.5 ± 0.2 × 107 m-1 s-1, respectively. Sequence analyses and a 1.9 A resolution crystal structure established that LigY belongs to the amidohydrolase superfamily, unlike previously characterized MCP hydrolases, which are serine-dependent enzymes of the α/β-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-β-carboxymuconic-ϵ-semialdehyde decarboxylase, a class III amidohydrolase, with a single zinc ion coordinated by His-6, His-8, His-179, and Glu-282. Interestingly, we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover, substitution of His-223, a conserved residue proposed to activate water in other amidohydrolases, reduced the kcat to a much lesser extent than what has been reported for other amidohydrolases, suggesting that His-223 has a different role in LigY. Substitution of Arg-72, Tyr-190, Arg-234, or Glu-282 reduced LigY activity over 100-fold. On the basis of these results, we propose a catalytic mechanism involving substrate tautomerization, substrate-assisted activation of water for hydrolysis, and formation of a gem-diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C-C-hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.

Lignostilbene-α,β-dioxygenase A (LsdA) from the bacterium Sphingomonas paucimobilis TMY1009 is a nonheme iron oxygenase that catalyzes the cleavage of lignostilbene, a compound arising in lignin transformation, to two vanillin molecules. To examine LsdA's substrate specificity, we heterologously produced the dimeric enzyme with the help of chaperones. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (k(cat)(app) = 1.00 ± 0.04 × 10(6) m(−1) s(−1)). These experiments further indicated that the substrate's 4-hydroxy moiety is required for catalysis and that this moiety cannot be replaced with a methoxy group. Phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene in a reversible, mixed fashion (K(ic) = 6 ± 1 μm, K(iu) = 24 ± 4 μm). An X-ray crystal structure of LsdA at 2.3 Å resolution revealed a seven-bladed β-propeller fold with an iron cofactor coordinated by four histidines, in agreement with previous observations on related carotenoid cleavage oxygenases. We noted that residues at the dimer interface are also present in LsdB, another lignostilbene dioxygenase in S. paucimobilis TMY1009, rationalizing LsdA and LsdB homo- and heterodimerization in vivo. A structure of an LsdA·phenylazophenol complex identified Phe(59), Tyr(101), and Lys(134) as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe(59) and Tyr(101) substitutions with His and Phe, respectively, reduced LsdA activity (k(cat)(app)) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys(134) plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.

A series of alkylated 2,3-dihydroxybiphenyls has been prepared on the gram scale by using an effective Directed ortho Metalation-Suzuki-Miyaura cross-coupling strategy. These compounds have been used to investigate the substrate specificity of the meta-cleavage dioxygenase BphC, a key enzyme in the microbial catabolism of biphenyl. Isolation and characterization of the meta-cleavage products will allow further study of related processes, including the catabolism of lignin-derived biphenyls.

In the catabolism of lignin-derived biphenyl by Sphingobium sp. SYK-6, LigZ catalyzes the cleavage of 2,2',3-trihydroxy-3'-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA) to a meta-cleavage product (MCP) identified here as 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (DCHM-HOPDA). DCHM-HOPDA is transformed nonenzymatically, likely to a lactone (k = 0.13 ± 0.01 min-1 , pH 7.5). This is hydrolyzed to the dienolate at alkaline pH (apparent pKa ~ 11.3). Only the dienolate is a substrate for LigY, the putative MCP hydrolase. LigZ has higher specificity for OH-DDVA (kcat /Km = 2.20 ± 0.02 × 107 s-1 ·m-1 ) than for protocatechuate (PCA; 6 ± 1 × 102 s-1 ·m-1 ). PCA also inactivates LigZ (partition ratio of 50), but at rates too low to be physiologically relevant. This study provides insight into the bacterial catabolism of lignin and facilitates the study of downstream catabolic enzymes.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes have wide catalytic versatility but are rarely known for their ability to react with oxygen to catalyze challenging reactions. Here, using in vitro reconstitution and kinetic analysis, we report that the indolmycin biosynthetic enzyme Ind4, from Streptomyces griseus ATCC 12648, is an unprecedented O2- and PLP-dependent enzyme that carries out a four-electron oxidation of L-arginine, including oxidation of an unactivated carbon-carbon (C-C) bond. We show that the conjugated product of this reaction, which is susceptible to nonenzymatic deamination, is efficiently intercepted and stereospecifically reduced by the partner enzyme Ind5 to give D-4,5-dehydroarginine. Thus, Ind4 couples the redox potential of O2 with the ability of PLP to stabilize anions to efficiently oxidize an unactivated C-C bond, with the subsequent stereochemical inversion by Ind5 preventing off-pathway reactions. Altogether, these results expand our knowledge of the catalytic versatility of PLP-dependent enzymes and enrich the toolbox for oxidative biocatalysis.