The missing link in linear alkylbenzenesulfonate surfactant degradation: 4-sulfoacetophenone as a transient intermediate in the degradation of 3-(4-sulfophenyl)butyrate by Comamonas testosteroni KF-1

Abstract

Biodegradation of the laundry surfactant linear alkylbenzenesulfonate (LAS) involves complex bacterial communities. The known heterotrophic community has two tiers. First, all LAS congeners are oxygenated and oxidized to about 50 sulfophenylcarboxylates (SPC). Second, the SPCs are mineralized. Comamonas testosteroni KF-1 mineralizes 3-(4-sulfophenyl)butyrate (3-C4-SPC). During growth of strain KF-1 with 3-C4-SPC, two transient intermediates were detected in the culture medium. One intermediate was identified as 4-sulfoacetophenone (SAP) (4-acetylbenzenesulfonate) by nuclear magnetic resonance (NMR). The other was 4-sulfophenol (SP). This information allowed us to postulate a degradation pathway that comprises the removal of an acetyl moiety from (derivatized) 3-C4-SPC, followed by a Baeyer-Villiger monooxygenation of SAP and subsequent ester cleavage to yield SP. Inducible NADPH-dependent SAP-oxygenase was detected in crude extracts of strain KF-1. The enzyme reaction involved transient formation of 4-sulfophenol acetate (SPAc), which was completely hydrolyzed to SP and acetate. SP was subject to NADH-dependent oxygenation in crude extract, and 4-sulfocatechol (SC) was subject to oxygenolytic ring cleavage. The first complete degradative pathway for an SPC can now be depicted with 3-C4-SPC: transport, ligation to a coenzyme A (CoA) ester, and manipulation to allow abstraction of acetyl-CoA to yield SAP, Baeyer-Villiger monooxygenation to SPAc, hydrolysis of the ester to acetate and SP, monooxygenation of SP to SC, the ortho ring-cleavage pathway with desulfonation, and sulfite oxidation.

FIG. 1.

Flow diagram of the primary degradation by P. lavamentivorans DS-1 of two LAS congeners to six SPCs, four of which are mineralized by C. testosteroni KF-1. None of the reactions or pathways indicated in strain DS-1 has been observed directly. None of the reactions in strain KF-1 has been observed directly, though 4-sulfophenol is a growth substrate (28): the major unknown in SPC degradation is the manipulation of the side chain.

FIG. 2.

Plot of (R)- and (S)-3-C4-SPC and sulfate concentrations versus cellular protein concentration during well-aerated growth of C. testosteroni KF-1 in 3-C4-SPC salts medium. The enantiomeric HPLC separation of (R)- and (S)-3-C4-SPC is given as an inset; no authentic standards of neither (R)- nor (S)-3-C4-SPC were available to assign identities to these peaks. Symbols: □, sulfate; ▵ and ▴, (R,S)-3-C4-SPC.

FIG. 3.

Growth of C. testosteroni KF-1 with 3-C4-SPC under oxygen-limited conditions (see the text) (A), and transient excretion of two degradation intermediates (B) identified as 4-sulfoacetophenone (SAP) (Fig. 4) and 4-sulfophenol (SP) (see the text). Symbols: ○, total protein; □, 3-C4-SPC; •, sulfate; ▵, SAP; ▴, SP.

FIG. 4.

Identification of 4-sulfoacetophenone by NMR. Shown are regions of interest of ¹H,¹³C-heteronuclear single quantum coherence (HSQC) (A) and HMBC (B) 2D NMR spectra recorded in CD₃OD solution (the regions with the resonances of the methyl group are not shown) with signal assignments (C) to the chemical structure of 4-sulfoacetophenone [4-acetylbenzene-sulfonate] isolated from the culture fluid.

FIG. 5.

NADPH-dependent transformation of SAP to SP with transient appearance of 4-sulfophenol acetate (A) and transformation of 4-sulfophenol acetate to SP and acetate (B). (A) A fraction of cell extract with SAP-oxygenase activity obtained from column-purification was used for the reaction; the initial SAP concentration was 0.3 mM. (B) Transformation of SPAc to SP and acetate was followed in cell extract. Symbols: □, SAP; •, 4-sulfophenol acetate (SPAc); ○, SP; ▵, acetate.

FIG. 6.

Postulated degradation pathway for (R, S)-3-C4-SPC and 3-C4-SPC-2H in C. testosteroni KF-1. The transport systems inferred from the ability of strain KF-1 to utilize (R,S)-3-C4-SPC, 3-C4-SPC-2H, SAP, SPAc, and SP as growth substrates, are also indicated. Enzymes: A, acyl-CoA synthetase (or acyl-CoA transferase?); B, acyl-CoA dehydrogenase; C, enoyl-CoA hydratase; D, keto-acid-lyase; E, Baeyer-Villiger-type monooxygenase; F, esterase; G, 4-sulfophenol 2-monooxygenase; H, 4-sulfocatechol 1,2-dioxygenase; I, 3-sulfomuconate cycloisomerase; J, 4-sulfomuconolactone hydrolase (desulfonating); K, maleylacetate reductase; L, succinyl-CoA:3-ketoadipate CoA-transferase; M, 3-ketoadipyl-CoA thiolase; N, sulfite dehydrogenase. Compounds: I, (R,S)-3-C4-SPC [(R,S)-3-(4-sulfophenyl)butyrate]; II, (R,S)-3-C4-SPC-CoA; III, 3-C4-SPC-2H [3-(4-sulfophenyl)-Δ2-enoyl-butyrate; 3-(4-sulfophenyl)crotonate]; IV, 3-C4-SPC-2H-CoA; V, 3-C4-SPC-OH-CoA [3-hydroxy-3-(4-sulfophenyl)butyryl-CoA]; VI, SAP (4-sulfoacetophenone); VII, SPAc (4-sulfophenol acetate); VIII, SP (4-sulfophenol); IX, SC (4-sulfocatechol); X, 3-sulfomuconate; XI, 4-sulfomuconolactone; XII, maleylacetate; XIII, 3-ketoadipate; and XIV, 3-ketoadipyl-CoA.