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Inhibition Mechanisms of Rhodococcus Erythropolis 2′-Hydroxybiphenyl-2-sulfinate Desulfinase (DszB)
journal contribution
posted on 2019-10-16, 17:33 authored by Yue Yu, Landon C. Mills, Derek L. Englert, Christina M. PayneNaturally occurring
enzymatic pathways enable highly specific,
rapid thiophenic sulfur cleavage occurring at ambient temperature
and pressure, which may be harnessed for the desulfurization of petroleum-based
fuel. One pathway found in bacteria is a four-step catabolic pathway
(the 4S pathway) converting dibenzothiophene (DBT), a common crude
oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the
carbon–carbon bonds. 2′-Hydroxybiphenyl-2-sulfinate
desulfinase (DszB), the rate-limiting enzyme in the
enzyme cascade, is capable of selectively cleaving carbon–sulfur
bonds. Accordingly, understanding the molecular mechanisms of DszB activity may enable development of the cascade as industrial
biotechnology. Based on crystallographic evidence, we hypothesized
that DszB undergoes an active site conformational
change associated with the catalytic mechanism. Moreover, we anticipated
this conformational change is responsible, in part, for enhancing
product inhibition. Rhodococcus erythropolis IGTS8 DszB was recombinantly produced and purified via Escherichia coli BL21 to test these hypotheses. Activity
and the resulting conformational change of DszB in
the presence of HBP were evaluated. The activity of recombinant DszB was comparable to the natively expressed enzyme and
was inhibited via competitive binding of the product, HBP. Using circular
dichroism, global changes in DszB conformation were
monitored in response to HBP concentration, which indicated that both
product and substrate produced similar structural changes. Molecular
dynamics (MD) simulations and free energy perturbation with Hamiltonian
replica exchange molecular dynamics (FEP/λ-REMD) calculations
were used to investigate the molecular-level phenomena underlying
the connection between conformation change and kinetic inhibition.
In addition to the HBP, MD simulations of DszB bound
to common, yet structurally diverse, crude oil contaminants 2′,2-biphenol
(BIPH), 1,8-naphthosultam (NTAM), 2-biphenyl carboxylic acid (BCA),
and 1,8-naphthosultone (NAPO) were performed. Analysis of the simulation
trajectories, including root-mean-square fluctuation (RMSF), center
of mass (COM) distances, and strength of nonbonded interactions, when
compared with FEP/λ-REMD calculations of ligand binding free
energy, showed excellent agreement with experimentally determined
inhibition constants. Together, the results show that the combination
of a molecule’s hydrophobicity and nonspecific interactions
with nearby functional groups contributes to a competitive inhibition
mechanism that locks DszB in a closed conformation
and precludes substrate access to the active site.
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Keywords
crude oil contaminantresults showthiophenic sulfur cleavageBIPHconformation changeHamiltonian replica exchangeRhodococcus erythropolis IGTS 8 DszBInhibition MechanismsBCAinhibition constantsDszB activityDBTrate-limiting enzymeambient temperatureEscherichia coli BL 21ligand bindingroot-mean-square fluctuation4 S pathwayenzyme cascadesimulation trajectoriesHBP concentrationpetroleum-based fuelCOMsubstrate accessnonbonded interactionsNAPOFEPMD simulationsmolecular-level phenomenaNTAMinhibition mechanism2- hydroxybiphenylDszB conformationenergy perturbationRMSFMolecular dynamicsproduct inhibitionfour-step catabolic pathwaylocks DszB
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