Anaerobic gram-negative bacterium Fusobacterium nucleatum (F. nucleatum) has long been found to cause opportunistic infections and has recently been implicated in colorectal cancer. In F. nucleatum, YggS (FnYggS) is an important part of bacterial cell walls and belongs to the COG0325 gene family. In this study, YggS from FnYggS was successfully expressed and purified using Ni-NTA affinity and gel-filtration chromatography. The protein crystal was obtained and diffracted to a resolution of 2.08 Å. The preliminary crystallographic analysis suggested that FnYggS crystal belongs to the monoclinic space group P2 1 with a = 37.93 Å, b = 146.38 Å, and c = 74.13 Å, α = γ = 90.00° and β = 93.36°. The asymmetric unit contained approximately three monomer of FnYggS, giving a crystal volume per mass (V M ) of 2.64 Å 3 Da -1 and a solvent content of 53.50%.
Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.
Catabolite
control protein E (CcpE) is a LysR-type transcriptional
regulator that positively regulates the transcription of the first
two enzymes of the TCA cycle, namely, citZ and citB, by sensing accumulated intracellular citrate. CcpE
comprises an N-terminal DNA-binding domain and a C-terminal regulatory
domain (RD) and senses citrate with conserved arginine residues in
the RD. Although the crystal structure of the apo SaCcpE-RD has been
reported, the citrate-responsive and DNA-binding mechanisms by which
CcpE regulates TCA activity remain unclear. Here, we report the crystal
structure of the apo and citrate-bound SaCcpE-RDs. The SaCcpE-RD exhibits
conformational changes between the two subdomains via hinge motion
of the central β4 and β10 strands. The citrate molecule
is located in a positively charged cavity between the two subdomains
and interacts with the highly conserved Ser98, Leu100, Arg145, and
Arg256 residues. Compared with that of the apo SaCcpE-RD, the distance
between the two subdomains of the citrate-bound SaCcpE-RD is more
than ∼3 Å due to the binding of the citrate molecule,
and this form exhibits a closed structure. The SaCcpE-RD exhibits
various citrate-binding-independent conformational changes at the
contacting interface. The SaCcpE-RD prefers the dimeric state in solution,
whereas the SaCcpE-FL prefers the tetrameric state. Our results provide
insight into the molecular function of SaCcpE.
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