A novel hydrolase identified by genomic-proteomic analysis of phenylurea herbicide mineralization by Variovorax sp. strain SRS16.
ABSTRACT The soil bacterial isolate Variovorax sp. strain SRS16 mineralizes the phenylurea herbicide linuron. The proposed pathway initiates with hydrolysis of linuron to 3,4-dichloroaniline (DCA) and N,O-dimethylhydroxylamine, followed by conversion of DCA to Krebs cycle intermediates. Differential proteomic analysis showed a linuron-dependent upregulation of several enzymes that fit into this pathway, including an amidase (LibA), a multicomponent chloroaniline dioxygenase, and enzymes associated with a modified chlorocatechol ortho-cleavage pathway. Purified LibA is a monomeric linuron hydrolase of ∼55 kDa with a K(m) and a V(max) for linuron of 5.8 μM and 0.16 nmol min⁻¹, respectively. This novel member of the amidase signature family is unrelated to phenylurea-hydrolyzing enzymes from Gram-positive bacteria and lacks activity toward other tested phenylurea herbicides. Orthologues of libA are present in all other tested linuron-degrading Variovorax strains with the exception of Variovorax strains WDL1 and PBS-H4, suggesting divergent evolution of the linuron catabolic pathway in different Variovorax strains. The organization of the linuron degradation genes identified in the draft SRS16 genome sequence indicates that gene patchwork assembly is at the origin of the pathway. Transcription analysis suggests that a catabolic intermediate, rather than linuron itself, acts as effector in activation of the pathway. Our study provides the first report on the genetic organization of a bacterial pathway for complete mineralization of a phenylurea herbicide and the first report on a linuron hydrolase in Gram-negative bacteria.
Article: Degradation of linuron and some other herbicides and fungicides by a linuron-inducible enzyme obtained from Bacillus sphaericus.[show abstract] [hide abstract]
ABSTRACT: Linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea] induces the formation of an enzyme (acylamidase) responsible for the degradation of a large variety of different herbicides and fungicides of the acylanilide and phenylurea type. The former type is degraded at a rate at least 10 times higher than the latter.Applied microbiology 10/1971; 22(3):284-8.
Article: Induction characteristics of reductive dehalogenation in the ortho-halophenol-respiring bacterium, Anaeromyxobacter dehalogenans.[show abstract] [hide abstract]
ABSTRACT: Anaeromyxobacter dehalogenans strain 2CP-C dehalogenates ortho-substituted di- and mono-halogenated phenols and couples this activity to growth. Reductive dehalogenation activity has been reported to be inducible, however, this process has not been studied extensively. In this study, the induction of reductive dehalogenation activity by strain 2CP-C is characterized. Constitutive 2-chlorophenol dechlorination activity occurs in non-induced fumarate-grown cells, with rates averaging 0.138 micromol of Cl- h(-1) mg of protein(-1). Once induced, these cultures dechlorinate 2- chlorophenol (2-CP) at rates as high as 116 micromol of Cl(-1) h(-1) mg of protein(-1). Dechlorination of 2-CP is induced by phenol, 2-chlorophenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 2,6-dichlorophenol, and 2-bromophenol. Of the substrates tested, 2-bromophenol shows the highest induction potential, yielding double the 2-chlorophenol dechlorination rate when compared to other inducing substrates. No induced dechlorination is observed at concentrations less than 5 microM 2-CP. When fumarate cultures were diluted 100-fold, fumarate reduction rates were reduced roughly according to the dilution factor, while dechlorination rates were similar in fumarate grown cells amended with 2-CP and cells diluted 100-fold prior to the addition of chlorophenol. This indicates that the majority of the fumarate-grown cells in late log phase were not induced when exposed to inducing substrates such as 2-CP. This observation may have ramifications on the success of bioaugmentation using halorespiring bacteria, which traditionally relies on growing cultures using more readily utilized substrates. The rapid dechlorination rate and unique induction pattern also make strain 2CP-C a promising model organism for understanding the regulation of reductive dehalogenation at the enzymatic level.Biodegradation 02/2002; 13(5):307-16. · 2.02 Impact Factor
Article: Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia.[show abstract] [hide abstract]
ABSTRACT: The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene ( pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His(6) tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala- L-Phe-NH(2) is hydrolyzed in the absence of cofactors to L-Ala- L-Phe-OH and ammonia with V(max)=194 U/mg and K(m) <0.5 mM. The natural function of Pam remains unclear. Chymostatin ( K(i)<0.3 microM) and Pefabloc SC ( K(i) not determined) were identified as inhibitors. When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S. maltophilia. In the expression system, Pam made up about 31% of the total soluble cell protein. From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.Applied Microbiology and Biotechnology 05/2002; 58(6):772-80. · 3.42 Impact Factor