Characterization of a Two-component Alkanesulfonate Monooxygenase from Escherichia coli

Abstract

The Escherichia coli ssuEADCB gene cluster is required for the utilization of alkanesulfonates as sulfur sources, and is expressed under conditions of sulfate or cysteine starvation. The SsuD and SsuE proteins were overexpressed and characterized. SsuE was purified to homogeneity as an N-terminal histidine-tagged fusion protein. Native SsuE was a homodimeric enzyme of M r 58,400, which catalyzed an NAD(P)H-dependent reduction of FMN, but it was also able to reduce FAD or riboflavin. The SsuD protein was purified to >98% purity using cation exchange, anion exchange, and hydrophobic interaction chromatography. The pure enzyme catalyzed the conversion of pentanesulfonic acid to sulfite and pentaldehyde and was able to desulfonate a wide range of sulfonated substrates including C-2 to C-10 unsubstituted linear alkanesulfonates, substituted ethanesulfonic acids and sulfonated buffers. SsuD catalysis was absolutely dependent on FMNH 2 and oxygen, and was maximal for SsuE/SsuD molar ratios of 2.1 to 4.2 in 10 mM Tris-HCl, pH 9.1. Native SsuD was a homotetrameric enzyme of M r 181,000. These results demonstrate that SsuD is a broad range FMNH 2 -dependent monooxygenase catalyzing the oxygenolytic conversion of alkanesulfonates to sulfite and the corresponding aldehydes. SsuE is the FMN reducing enzyme providing SsuD with FMNH 2 .In Escherichia coli, sulfate starvation causes increased synthesis of several proteins involved in scavenging sulfur from alternative sulfur sources (1). Among these proteins are the tauABCD-encoded proteins required for uptake and desulfonation of taurine (2-aminoethanesulfonic acid) (2, 3) and the proteins SsuE and SsuD of the ssuEADCB gene cluster. We have shown that the ssuEADCB gene cluster, located at 21.4 min on the E. coli chromosome, is specifically involved in the utilization of alkanesulfonates as a source of sulfur for growth (4). Deletion of ssuEADCB resulted in the loss of the ability to utilize alkanesulfonates as a sulfur source but did not affect the utilization of taurine for this purpose. The amino acid sequences of SsuABC exhibit similarity to components of ABCtype transport systems (4, 5). SsuA has a putative signal sequence, indicating that it functions as a periplasmic binding protein, and the sequences of SsuB and SsuC are significantly similar to those of ATP-binding proteins and membrane components, respectively, of members of the ABC transporter superfamily. It thus appears that the proteins encoded by ssuABC constitute an uptake system for alkanesulfonates.The ssuD gene product shows 25% sequence identity to a characterized nitrilotriacetate two-component monooxygenase of Chelatobacter heintzii (6) and to the pristinamycin II A synthase subunit A of Streptomyces pristinaespiralis (7), suggesting that SsuD is involved in the oxygenolytic release of sulfite from alkanesulfonates. Here we report the purification of the SsuD and SsuE proteins, describe their biochemical properties, and demonstrate that SsuD is a monooxygenase that catalyzes the desulfonation of alkanesulf...

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

“…For example, the apparent specificity (k cat /K m ) of a bacterial EDTA monooxygenase for MgEDTA 2Ϫ is 430-fold greater than that of HsaAB (51). Simi- larly, the apparent specificity of alkanesulfonate monooxygenase for alkanesulfonates was up to 112-fold greater (25). Nevertheless, the relative activity of HsaAB is very similar to that of its flanking enzymes in the cholesterol catabolic pathway, KshA (17) ), respectively, would help minimize intracellular concentrations of the latter, which is toxic to Mtb (6).…”

“…Examples for the former include 4-hydroxyphenyl acetate 3-monooxygenase (HpaB) of Escherichia coli (21) and 2,4,5-trichlorophenol monooxygenase from Burkholderia cepacia (22). FMN-dependent systems are involved in the synthesis of antibiotics such as actinorhodin in Streptomyces coelicolor (23), the bacterial degradation of polyaminocarboxylates such as nitrilotriacetic acid (24), and the desulfurization of fossil fuels by rhodococci (25). pHPAH (EC 1.14.13.3), a well characterized FMN-utilizing enzyme from Acinetobacter baumanii, catalyzes the hydroxylation of p-hydroxyphenyl acetate (HPA) to 3,4-dihydroxyphenyl acetate (PDB entry 2JBT) (26).…”

The Peroxiredoxin Repair Proteins

“…Until recently these cysteine derivatives were viewed as irreversible; although sulfinic acids can be reduced in vitro by reductants such as 2-mercaptoethanol under very acidic (< pH 4) conditions (Finlayson, 1979;Claiborne et al, 1999). Interestingly, bacteria have enzymatic systems, such as the E. coli SsuE and SsuD, that during sulfate starvation scavenge sulfur from alkane sulfonates to generate the corresponding aldehyde and sulfite (Eichhorn et al, 1999).…”

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

“…2B). Furthermore, the product coeluted with an authentic standard of 7-chlorotryptophan (23 In the absence of RebF, RebH showed low-level chlorinating activity that may have resulted from low-level contamination by highly active E. coli flavin reductases (26,27) during heterologous expression and purification. However, rates of product formation were dramatically enhanced with addition of RebF.…”