Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase

https://doi.org/10.1016/j.bbrc.2016.10.145Get rights and content

Highlights

  • Dibenzothiophene monooxygenase forms and stabilizes a C4a-peroxyflavin intermediate.

  • S163 and H391 are required for stabilization of the peroxyflavin intermediate.

  • H391's positive charge is likely involved in catalyzing the flavin - oxygen reaction.

  • S163 likely stabilizes the peroxyflavin to elimination of hydrogen peroxide.

Abstract

Dibenzothiophene monooxygenase is the initiating enzyme in the Rhodococcus 4S biodesulfurization pathway. A member of the Class D flavin monooxygenases, it uses FMN to activate molecular oxygen for oxygenation of the substrate, dibenzothiophene. Here, we have used stopped-flow spectrophotometry to show that DszC forms a peroxyflavin intermediate in the absence of substrate. Mutagenesis of Ser163 and His391 to Ala appears to decrease the binding affinity for reduced FMN and eliminates the enzyme's ability to stabilize the peroxyflavin intermediate.

Introduction

SO2 is an atmospheric pollutant generated by both natural and industrial processes, including combustion of organosulfur compounds found as contaminants in fuel. Hydrodesulfurization, the current method of sulfur removal [1], can remove most simple sulfur-containing contaminants, but certain aromatic compounds (refractory organosulfur compounds) cannot be effectively treated this way [2]. Several bacterial species are capable of removing sulfur from specific refractory organosulfur compounds such as dibenzothiophene (DBT) [3], [4]. In Rhodococcus erythropolis, a three-enzyme pathway (DszCAB) removes sulfur from DBT to produce hydroxybiphenyl and sulfite for metabolism [5], [6], [7]. A detailed understanding of catalysis by DszCAB would prove invaluable toward development of biodesulfurization for industrial purposes.

DszC is the monooxygenase of a two-component flavin monooxygenase system that catalyzes two sequential S-oxidations of DBT to dibenzothiophene 5-sulfoxide (DBTO) and then dibenzothiophene 5,5-dioxide (DBTO2, Supplemental Fig. 1). The enzyme uses reduced flavin mononucleotide (FMNH) to activate molecular oxygen for reaction with the sulfenyl (or sulfinyl) sulfur of DBT (or DBTO) [5], possibly by electrophilic oxidation involving the known flavin-oxygen adduct, C4a-hydroperoxyflavin [8]. The FMNH required for catalysis is produced by DszD [9], the flavin reductase component of the two-component monooxygenase system [10]. Five X-ray crystal structures have shown DszC to be a tetrameric protein with sequence and structural similarity to the acyl-CoA dehydrogenases [8], [11], [12], [13]. Based on its sequence homology and catalytic requirements, DszC is a Class D monooxygenase [14], [15], along with p-hydroxyphenylacetate hydroxylase (HpaH) [16], chlorophenol-4-monooxygenase [17], nitrososynthase [18], and others [19], [20], [21], [22]. DszC interacts with flavin as do other Class D monooxygenases [8], [11], [12], [13], and two loops adjacent to the active site, both of which adopt distinct “open” and “closed” conformations, may control access to the active site pocket [8], [13]. Mutagenesis of individual active site residues, including H92, S163, H388, H391 and D392 (Supplemental Fig. 2), abolishes enzymatic activity [8], [12]. The specific catalytic roles of each of these residues are currently unknown.

Here, we begin our study of catalysis by DszC and the functional roles of two residues in the DszC active site, S163 and H391. Based on studies of HpaH, S163 and H391 are likely to be involved in formation of C4a-hydroperoxyflavin [23]. H391 is suspected to facilitate reaction with O2 by decreasing the kinetic barrier to electron transfer to O2 by protonating the C4a-peroxyflavin anion [8], [23], [24], while S163 provides stabilizing interactions to the peroxyflavin through a hydrogen bond to N5 of the isoalloxazine [23]. To investigate these proposals, both residues were mutated to alanine and studied in comparison to wild-type DszC by steady state activity assays and stopped-flow kinetics to examine the reaction of reduced DszC with O2.

Section snippets

Cloning and purification

The codon-optimized Rhodococcus erythropolis DszC gene was obtained from GenScript USA Inc. (Piscataway, NJ) in pUC57 and subcloned into the E. coli expression vector pBG100 (Vanderbilt University's Center for Structural Biology), which confers a cleavable N-terminal 6-histidine tag and kanamycin resistance. The S163A- and H391A-DszC mutant plasmids were created using the Phusion Site-Directed Mutagenesis Kit (Thermo) and custom primers (Supplemental Table 1). DNA was propagated in E. coli DH5α

Cloning, mutagenesis, purification and analysis of wild-type and mutant DszC

The dszC gene was subcloned into plasmid pBG100 and verified by DNA sequencing (Supplemental Information). S163A-DszC and H391A-DszC were made by site-directed mutagenesis and were also confirmed by sequencing. Wild-type and mutant DszC expressed at high levels and were purified to apparent homogeneity using nickel affinity chromatography (Supplemental Fig. 3) at yields of 60–70 mg of purified protein per liter of culture. Native PAGE (Supplemental Fig. 4) and size exclusion chromatography

Acknowledgements

The authors acknowledge Aparna Sapra for assistance during data collection. This work was supported by the Research Corporation [CCSA 22672] and the NIH [Grant 5SC2AI109500].

References (31)

Cited by (6)

  • Flavin-N5-oxide intermediates in dibenzothiophene, uracil, and hexachlorobenzene catabolism

    2019, Methods in Enzymology
    Citation Excerpt :

    The search for a microbial desulfurization process has resulted in the identification of a dibenzothiophene catabolic pathway in Rhodococcus erythropolis (Fig. 3) (Denome, Oldfield, Nash, & Young, 1994; Denome, Olson, & Young, 1993; Gray et al., 1996; Piddington, Kovacevich, & Rambosek, 1995). The pathway involves thioether oxidation catalyzed by the DszC (Barbosa, Neves, Sousa, Ramos, & Fernandes, 2018; Gonzalez-Osorio, Luong, Jirde, Palfey, & Vey, 2016; Guan et al., 2015) flavoenzyme, oxidative C–S bond cleavage catalyzed by the DszA flavoenzyme and a final C–S bond cleavage catalyzed by DszB (Lee, Ohshiro, Matsubara, Izumi, & Tanokura, 2006). The DszA catalyzed C–S bond cleavage is a novel reaction meriting further mechanistic exploration.

  • Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity

    2017, Journal of Biological Chemistry
    Citation Excerpt :

    DszC contains His-92 and Ser-163 at almost the same positions as His-89 and Ser-160 in TdsC. Both mutations H92F (21) and S163A (21, 34) substantially reduced the DBT oxygenation activity of DszC, as found for the TdsC mutants H89A and S160A. The hydrogen-bonding network from the flavin N5 atom to Ser and aromatic side chains was also found in p-hydroxyphenylacetate hydroxylase C2.

1

Present address: School of Medicine, Vanderbilt University, Nashville, TN 27232, United States.

View full text