Cleavage of the ␤-aryl ether linkage is the most important process in lignin degradation. Here we characterize the three tandemly located glutathione S-transferase (GST) genes, ligF, ligE, and ligG, from lowmolecular-weight lignin-degrading Sphingomonas paucimobilis SYK-6, and we describe the actual roles of these genes in the ␤-aryl ether cleavage. Based on the identification of the reaction product by electrospray ionization-mass spectrometry, a model compound of ␤-aryl ether, ␣-(2-methoxyphenoxy)-␤-hydroxypropiovanillone (MPHPV), was transformed by LigF or LigE to guaiacol and ␣-glutathionyl-␤-hydroxypropiovanillone (GS-HPV). This result suggested that LigF and LigE catalyze the nucleophilic attack of glutathione on the carbon atom at the ␤ position of MPHPV. High-pressure liquid chromatography-circular dichroism analysis indicated that LigF and LigE each attacked a different enantiomer of the racemic MPHPV preparation. The ligG gene product specifically catalyzed the elimination of glutathione from GS-HPV generated by the action of LigF. This reaction then produces an achiral compound, ␤-hydroxypropiovanillone, which is further degraded by this strain. Disruption of the ligF, ligE, and ligG genes in SYK-6 showed that ligF is essential to the degradation of one of the MPHPV enantiomers, and the alternative activities which metabolize the substrates of LigE and LigG are present in this strain.

Burkholderia sp. strain TH2, a 2-chlorobenzoate (2CB)-degrading bacterium, metabolizes benzoate (BA) and 2CB via catechol. Two different gene clusters for the catechol ortho-cleavage pathway (cat1 and cat2) were cloned from TH2 and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis showed that while both catechol dioxygenases (CatA1 and CatA2) were produced in BA-grown cells, CatA1 was undetectable when strain TH2 was grown on 2CB or cis,cis-muconate (CCM), an intermediate of catechol degradation. However, production of CatA1 during growth on 2CB or CCM was observed when cat2 genes were disrupted. The difference in the production of CatA1 and CatA2 was apparently due to a difference in inducer recognition by the regulators of the gene clusters. The inducer of CatA1 was found to be BA, not 2CB, by using a 2-halobenzoate dioxygenase gene (cbd) disruptant, which is incapable of transforming (chloro)benzoate. It was also found that CCM or its metabolite acts as an inducer for CatA2. When cat2 genes were disrupted, the growth rate in 2CB culture was reduced while that in BA culture was not. These results suggest that although cat2 genes are not indispensable for growth of TH2 on 2CB, they are advantageous.Chlorobenzoates (CBs) are key intermediates in the degradative pathways of polychlorinated biphenyls (PCBs). Therefore, the degradation of CBs has been extensively studied in conjunction with that of PCBs. CBs degraded by aerobic bacteria are often converted to nonaromatic chloro-cyclohexadiene-1,2-diol-1-carboxylic acid (DHB) by (chloro)benzoate dioxygenase and then to chlorocatechols by DHB dehydrogenase (11). The chlorocatechols yielded are then catabolized via a modified ortho-cleavage pathway (21, 29, 32) or a metacleavage pathway (18,33). In 2-chlorobenzoate (2CB)-degrading bacteria, such as Burkholderia sp. strain TH2 (44) and Pseudomonas sp. strain 2CBS (9), degradation of 2CB is initiated by 2-halobenzoate dioxygenase (CbdABC) (13), giving rise to 2-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (2-Cl-DHB), which is presumed to spontaneously lose carbon dioxide and halogenide and generate catechol (10). Accordingly, DHB dehydrogenase is not necessary for 2CB degradation in these bacteria. Catechol degradation in these 2CB-degrading bacteria, however, has not been studied extensively at the genetic level.Burkholderia sp. strain TH2 has 2-halobenzoate dioxygenase genes (cbdABC) nearly identical to those of Pseudomonas sp. strain 2CBS (13, 44). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins induced when strain TH2 was grown on 2CB or benzoate (BA) showed that the N-terminal sequences of the induced proteins with molecular masses of 34 and 32 kDa were similar to those of catechol 1,2-dioxygenases of other bacteria (44), suggesting that strain TH2 has two catechol 1,2-dioxygenases. What is more interesting is that while either protein was induced during growth on BA, a protein with a molecular mass of 32 kDa was not obser...

Two distinct dipeptidyl aminopeptidases, which were designated DPP-A and DPP-B, were purified from soluble fraction of monkey brain using Leu-enkephalin as the substrate. The enzymes were purified 187 and 136 fold, respectively. Both enzymes showed the optimum pH in neutral range. Their molecular weights were almost equal and were estimated to be about 100,000. Their Km values with Leu-enkephalin as the substrate were 5.6 X 10(-5) and 1.1 X 10(-5) M, respectively. Among synthesized substrates, the highest affinity of the enzymes was toward arginyl-arginine beta-naphthylamide with the Km values of 6.25 X 10(-5) and 6.41 X 10(-5) M, respectively. Both enzyme activities were inhibited by the metal-chelators DFP and PCMB. Two hundred fifty microM arphamenine A inhibited DPP-A and -B with inhibition of 36.6% and 44.1%, respectively. Beta-endorphin, ACTH, and glucagon inhibited only DPP-B, while beta-lipotropin and angiotensin II inhibited both DPP-A and -B when Leu-enkephalin was used as the substrate.