Hexachlorobenzene Monooxygenase Substrate Selectivity and Catalysis: Structural and Biochemical Insights

Abstract

Hexachlorobenzene (HCB), as one of the persistent organic pollutants (POPs) and a possible human carcinogen, is especially resistant to biodegradation. In this study, HcbA1A3, a distinct flavin-N5-peroxide-utilizing enzyme and the sole known naturally occurring aerobic HCB dechlorinase, was biochemically characterized. Its apparent preference for HCB in binding affinity revealed that HcbA1 could oxidize only HCB rather than less-chlorinated benzenes such as pentachlorobenzene and tetrachlorobenzenes. In addition, the crystal structure of HcbA1 and its complex with flavin mononucleotide (FMN) were resolved, revealing HcbA1 to be a new member of the bacterial luciferase-like family. A much smaller substrate-binding pocket of HcbA1 than is seen with its close homologues suggests a requirement of limited space for catalysis. In the active center, Tyr362 and Asp315 are necessary in maintaining the normal conformation of HcbA1, while Arg311, Arg314, Phe10, Val59, and Met12 are pivotal for the substrate affinity. They are supposed to place HCB at a productive orientation through multiple interactions. His17, with its close contact with the site of oxidation of HCB, probably fixes the target chlorine atom and stabilizes reaction intermediates. The enzymatic characteristics and crystal structures reported here provide new insights into the substrate specificity and catalytic mechanism of HcbA1, which paves the way for its rational engineering and application in the bioremediation of HCB-polluted environments.IMPORTANCE As an endocrine disrupter and possible carcinogen to human beings, hexachlorobenzene (HCB) is especially resistant to biodegradation, largely due to difficulty in its dechlorination. The lack of knowledge of HCB dechlorinases limits their application in bioremediation. Recently, an HCB monooxygenase, HcbA1A3, representing the only naturally occurring aerobic HCB dechlorinase known so far, was reported. Here, we report its biochemical and structural characterization, providing new insights into its substrate selectivity and catalytic mechanism. This research also increases our understanding of HCB dechlorinases and flavin-N5-peroxide-utilizing enzymes.

Keywords: biodegradation; catalytic mechanism; crystal structure; dechlorinase; flavin-N5-peroxide; hexachlorobenzene; monooxygenase.

FIG 1

Comparisons of proposed metabolic pathways of HCB in anaerobic strain CBDB1 and aerobic strain PD653. TeCH, tetrachloro-p-hydroquinone; DiCH, dichloro-p-hydroquinone.

FIG 2

Biochemical characterization of HcbA1A3. (a) Effect of molar ratios of HcbA1 to HcbA3 on hexachlorobenzene (HCB) consumption. (b) Effect of reaction duration on HCB consumption. The ordinate shows the percentages of residual HCB with respect to the initial HCB level. (c) Effect of temperature on the enzyme activity of HcbA1A3. (d) Effect of pH on the enzyme activity of HcbA1A3. The gray line represents citrate buffer, the blue line represents MES buffer, the red line represents HEPES buffer, the orange line represents Tris-HCl buffer, and the green line represents glycine buffer.

FIG 3

The structure of HcbA1. (a) Overall structure of the HcbA1 dimer. Each subunit is shown in a different color (chain A, blue; chain B, green). (b) Five insertions (AI1 to AI5) of the HcbA1 subunit. AI1, AI2, AI3, AI4, AI5, and the C-extension are colored in wheat, yellow, green, cyan, pink, and blue, respectively. (c) F_o-F_c difference map calculated with the ligand omitted, contoured at 2.0 σ around modeled FMN. The hydrogen bond interactions of FMN with surrounding residues are shown as gray dashes. (d) The surface of HcbA1 features a substrate and flavin binding pocket. (e) The structure model of HcbA1·FMN docked with hexachlorobenzene (HCB). The orange dashed lines show the distances from the site of oxidation to the reactive N5 of FMN and from the target chlorine atom to the Nε2 of His17. The hydrogen bond network of the active center is shown as gray dashes. (f) A schematic of the active site of HcbA1.

FIG 4

The interatomic distances measured during molecular dynamics (MD) simulations of HcbA1·FMN·HCB. During the MD simulations, C1 and Cl1 of HCB showed shorter distances with respect to N5/C4a of FMN than with those of C2 and Cl2. (a) Distances between C1 and N5/C4a. (b) Distances between C2 and N5/C4a. (c) Distances between Cl1 and N5/C4a. (d) Distances between Cl2 and N5/C4a. The distances shown here represent the statistics for 50 to 150 ns during MD.

FIG 5

Substrate-enzyme binding affinity analysis by ForteBio Octet. (a) HCB, PCB, 1,2,3,4-TeCB, and 1,2,4,5-TeCB were used to test their binding affinities with wild-type HcbA1. (b) HCB was used to test its binding affinities with wild-type HcbA1, as well with as its mutants H17A, M12A, R311L, R314L, H79A, V59A, and F10A.

FIG 6

Structural comparison of HcbA1 and its homologues. (a) Superposition of HcbA1 (green), LadA (blue), and EmoA (orange). (a-1) Comparison between HcbA1 (green) and LadA (blue) in segments AI2 and AI5. (a-2) Comparison between HcbA1 (green) and EmoA (orange) in segments AI2 and AI5. (b) Superposition of HcbA1 (green) and RutA (pink). (b-1) Comparison between the substrate-binding pockets of HcbA1 (green) and RutA (pink).