Investigation of active site amino acid influence on carbon and chlorine isotope fractionation during reductive dechlorination

Abstract

Reductive dehalogenases (RDases) are corrinoid-dependent enzymes that reductively dehalogenate organohalides in respiratory processes. By comparing isotope effects in biotically-catalyzed reactions to reference experiments with abiotic corrinoid-catalysts, compound-specific isotope analysis (CSIA) has been shown to yield valuable insights into enzyme mechanisms and kinetics, including RDases. Here, we report isotopic fractionation (ε) during biotransformation of chloroform (CF) for carbon (εC = -1.52 ± 0.34‰) and chlorine (εCl = -1.84 ± 0.19‰), corresponding to a ΛC/Cl value of 1.13 ± 0.35. These results are highly suppressed compared to isotope effects observed both during CF biotransformation by another organism with a highly similar RDase (> 95% sequence identity) at the amino acid level, and to those observed during abiotic dehalogenation of CF. Amino acid differences occur at four locations within the two different RDases’ active sites, and this study examines whether these differences potentially affect the observed εC, εCl, and ΛC/Cl. Structural protein models approximating the locations of the residues elucidate possible controls on reaction mechanisms and/or substrate binding efficiency. These four locations are not conserved among other chloroalkane reducing RDases with high amino acid similarity (> 90%), suggesting that these locations may be important in determining isotope fractionation within this homologous group of RDases.

Respiratory protein interactions in Dehalobacter sp. strain 8M revealed through genomic and native proteomic analyses

“…HupL and HupS had identical migration patterns, indicating firm interaction (Figures 3, S5 and S6), as has F I G U R E 5 Alignment of the RdhA region has been suggested to determine isotopic fractionation of TCM in different OHRBs (Phillips et al, 2022). Highlighted residues indicate differences from the consensus sequence.…”

“…This difference in the isotopic pattern has led to questions about the reaction mechanisms of very similar RdhA proteins. In a recent study, Phillips et al (2022) hypothesized that these differences in carbon and chlorine isotope effects are related to amino acid differences in the active sites of CfrA compared to TmrA and the KB-1 Plus CF RdhA. They proposed that the low C and Cl isotope effects observed in cultures containing TmrA and the KB-1 Plus CF RdhA are associated with the similarity in their amino acid residues Phe80, Phe388 and Trp391.…”

“…strain 8M enrichment compared to other Dehalobacter ‐containing enrichments (Heckel et al, 2019; Soder‐Walz et al, 2022). This phenomenon has recently been attributed to amino acid differences in the vicinity of the active site of the TCM‐transforming RdhA (Phillips et al, 2022).…”

A Multifunctional Dehalobacter? Tandem Chloroform and Dichloromethane Degradation in a Mixed Microbial Culture

“…The original SC05 culture (also known as KB-1 Plus CF) has been maintained since 2010 at SiREM (Guelph, ON) in defined FeS-reduced, bicarbonate-buffered mineral medium amended weekly with CF as the electron acceptor and methanol (1 mM) and ethanol (0.5 mM) as electron donors, each at an electron equivalency of 6× CF demand [2 electron equivalents (eeq) per mole of CF]. A culture aliquot was provided to the University of Toronto in 2018, referred to herein as SC05-UT, where it was initially amended with CF, ethanol, and lactate . Subsequently, the culture was only refed CFwithout an electron donordue to the work by Wang et al showing that hydrogen production from DCM metabolism might be able to fuel CF dechlorination without exogenous donor addition …”

“…A culture aliquot was provided to the University of Toronto in 2018, referred to herein as SC05-UT, where it was initially amended with CF, ethanol, and lactate. 21 Subsequently, the culture was only refed CF�without an electron donor�due to the work by Wang et al showing that hydrogen production from DCM metabolism might be able to fuel CF dechlorination without exogenous donor addition. 19 After maintenance for more than one year without electron donor amendment, a subsample of SC05-UT was inoculated into fresh medium (day 0, Figure 2), which was named "DCME" (for DCM enrichment) and henceforth maintained by the addition of DCM alone.…”