Nitroaromatic compounds are typically toxic and resistant to degradation. Bradyrhizobium species strain JS329 metabolizes 5-nitroanthranilic acid (5NAA), a metabolite secreted by Streptomyces scabies, the bacterium responsible for potato scab, an agricultural disease of global economic importance. The first biodegradation enzyme is 5NAA-aminohydrolase (5NAA-A), a metalloprotease family member that converts 5NAA to 5-nitrosalicylic acid. We characterized 5NAA-A biochemically and obtained snapshots of its mechanism. 5NAA-A, an octamer that can use several divalent transition metals for catalysis, employs a nucleophilic aromatic substitution mechanism. Unexpectedly, the metal in 5NAA-A is labile but readily loaded in the presence of substrate. 5NAA-A is specific for 5NAA and cannot hydrolyze other tested derivatives, which are likewise poor inhibitors. The 5NAA-A structure and mechanism expands our understanding of the chemical ecology of an agriculturally important plant and pathogen, and will inform bioremediation and biocatalytic approaches to mitigate the environmental and ecological impact of nitroanilines and other challenging substrates.
Dihydroneopterin triphosphate pyrophosphatase (DHNTPase), a member of the Mg2+ dependent Nudix hydrolase superfamily, is the recently-discovered enzyme that functions in the second step of the pterin branch of the folate biosynthetic pathway in E. coli. DHNTPase is of interest because inhibition of enzymes in bacterial folate biosynthetic pathways is a strategy for antibiotic development. We determined crystal structures of DHNTPase with and without activating, Mg2+-mimicking metals Co2+ and Ni2+. Four metal ions, identified by anomalous scattering, and stoichiometrically confirmed in solution by isothermal titration calorimetry, are held in place by Glu56 and Glu60 within the Nudix sequence motif, Glu117, waters, and a sulfate ion, of which the latter is further stabilized by a salt bridge with Lys7. In silico docking of the DHNTP substrate reveals a binding mode in which the pterin ring moiety is nestled in a largely hydrophobic pocket, the β-phosphate activated for nucleophilic attack overlays with the crystallographic sulfate and is in line with an activated water molecule, and remaining phosphate groups are stabilized by all four identified metal ions. The structures and binding data provide new details regarding DHNTPase metal requirements, mechanism, and suggest a strategy for efficient inhibition.
KCNH2
encodes hERG1, the voltage-gated potassium channel that conducts the rapid delayed rectifier potassium current (I
Kr
) in human cardiac tissue. hERG1 is one of the first channels expressed during early cardiac development, and its dysfunction is associated with intrauterine fetal death, sudden infant death syndrome, cardiac arrhythmia, and sudden cardiac death. Here, we identified a hERG1 polypeptide (hERG1
NP
) that is targeted to the nuclei of immature cardiac cells, including human stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat cardiomyocytes. The nuclear hERG1
NP
immunofluorescent signal is diminished in matured hiPSC-CMs and absent from adult rat cardiomyocytes. Antibodies targeting distinct hERG1 channel epitopes demonstrated that the hERG1
NP
signal maps to the hERG1 distal C-terminal domain.
KCNH2
deletion using CRISPR simultaneously abolished I
Kr
and the hERG1
NP
signal in hiPSC-CMs. We then identified a putative nuclear localization sequence (NLS) within the distal hERG1 C-terminus, 883-RQRKRKLSFR-892. Interestingly, the distal C-terminal domain was targeted almost exclusively to the nuclei when overexpressed HEK293 cells. Conversely, deleting the NLS from the distal peptide abolished nuclear targeting. Similarly, blocking α or β1 karyopherin activity diminished nuclear targeting. Finally, overexpressing the putative hERG1
NP
peptide in the nuclei of HEK cells significantly reduced hERG1a current density, compared to cells expressing the NLS-deficient hERG1
NP
or GFP. These data identify a developmentally regulated polypeptide encoded by
KCNH2
, hERG1
NP
, whose presence in the nucleus indirectly modulates hERG1 current magnitude and kinetics.
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