Two Enzymes of a Complete Degradation Pathway for Linear Alkylbenzenesulfonate (LAS) Surfactants: 4-Sulfoacetophenone Baeyer-Villiger Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1

Abstract

Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C 4 -SPC). Second, these SPCs are mineralized. 3-C 4 -SPC is mineralized by Comamonas testosteroni KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C 4 -SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria.

Permanent draft genome sequence of Comamonas testosteroni KF-1

“…The 3-C 4 -SPC is converted to SAP [9] and further to 4-sulfophenol acetate (SPAc) by a recently identified Baeyer-Villiger monooxygenase (‘SAPMO’, PD5437), and SPAc hydrolyzed by a recently identified carboxylester hydrolase encoded by the next gene in the genome (PD5438), to yield acetate and SP [10]. The two identified genes, together with other (predicted) catabolic genes, are framed by IS 1071 insertion sequence elements (Tn 3 -family transposase genes), which suggests that these genes have only recently been acquired, possibly in the form of a ‘catabolic composite transposon’ through horizontal gene transfer [10]. Genes for other sections of the proposed 3-C 4 -SPC degradation pathway in strain KF-1, i.e., the ‘upper’ and ‘lower’ pathway, from 3-C 4 -SPC to SAP and from SP further to central metabolites, respectively [9], are examined in our present work (unpublished).…”

“…In particular, strain KF-1 was isolated from a laboratory trickling filter that had been used to enrich a bacterial community from sewage sludge that completely degraded commercial LAS and SPCs [1,6]. Strain KF-1 is able to utilize four individual SPCs (both enantiomers), namely R / S -3-(4-sulfopenyl)butyrate (3-C 4 -SPC), enoyl-3-C 4 -SPC, R / S -3-(4-sulfopenyl)pentanoate (3-C 5 -SPC), and enoyl-3-C 5 -SPC (see therefore also below), as novel carbon an energy sources for its heterotrophic aerobic growth [1,9,10]. …”

“…In several C. testosteroni strains, the physiology, biochemistry, genetics, and/or regulation of the utilization of aromatic compounds have been elucidated [e.g., 10,23,25,27,29,32-48]. Furthermore, the genome sequence of (plasmid-cured) C.…”

Entner–Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1

“…Two-dimensional PAGE was performed using Bio-Rad's ReadyStrip immobilized pH gradient system (36). For RT-PCR assays (primer sequences; Table S1), the E.Z.N.A Bacterial RNA preparation kit (Omega Bio-Tek) was used, and for complementary cDNA synthesis, the Maxima reverse transcriptase kit (Fermentas) was used; Taq polymerase (Fermentas) was used for subsequent PCR assays (37). For insertional mutagenesis via homologous recombination, an internal 500-bp sequence of gene 0090 cloned into vector PCR2.1 (Invitrogen) was used; the construct was introduced by electroporation, and mutants were selected on kanamycin LBagar plates.…”

Substitution of a Single Amino Acid Reverses the Regiospecificity of the Baeyer–Villiger Monooxygenase PntE in the Biosynthesis of the Antibiotic Pentalenolactone

“…The first example of a biological Baeyer-Villiger reaction catalyzed by a Baeyer-Villiger monooxygenase (BVMO) was reported in 1948. 1 Since then, many BVMOs have been isolated from a variety of microorganisms, typically associated with catabolic pathways, including cyclopentanone monooxygenase (CPMO), 2 cyclohexanone monooxygenase (CHMO), 3 cyclododecanone monooxygenase (CDMO), 4 cyclopentadecanone monooxygenase (CPDMO), 5 4-hydroxyacetophenone monooxygenase (HAPMO), 6 4-sulfoacetophenone monooxygenase (SAPMO), 7 phenylacetone monooxygenase (PAMO), 8 and steroid monooxygenase (SMO). 9 More recently, the first examples of BVMOs involved in anabolic pathways have been reported.…”