A phospho‐sugar binding domain homologous to NagB enzymes regulates the activity of the central glycolytic genes repressor

Abstract

CggR belongs to the SorC family of bacterial transcriptional regulators which control the expression of genes and operons involved in carbohydrate catabolism. CggR was first identified in Bacillus subtilis where it represses the gapA operon encoding the five enzymes that catalyze the central part of glycolysis. Here we present a structure/function study demonstrating that the C-terminal region of CggR regulates the DNA binding activity of this repressor in response to binding of a phosphorylated sugar. Molecular modeling of CggR revealed a winged-helix DNA-binding motif followed by a C-terminal domain presenting weak but significant homology with glucosamine-6-phosphate deaminases from the NagB family. In silico ligand screening suggested that the CggR C-terminal domain would bind preferentially bi-phosphorylated compounds, in agreement with previous studies that proposed fructuose-1,6-biphosphate (FBP) as the inducer metabolite. In vitro, FBP was the only sugar compound capable of interfering with CggR cooperative binding to DNA. FBP was also found to protect CggR against trypsin degradation at two arginine residues predicted to reside in a mobile loop forming the active site lid of the NagB enzymes. Replacement of residues predicted to interact with FBP led to mutant CggR with altered repressor activity in vivo but retaining their structural integrity and DNA binding activity in vitro. Interestingly, some of the mutant repressors responded with different specificity towards mono- and di-phospho-fructosides. Based on these results, we propose that the activity of the CggR-like repressors is controlled by a phospho-sugar binding (PSB) domain presenting structural and functional homology with NagB enzymes.

Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex

“…These observations are in good agreement with our previous studies on CggR/DNA interactions by fluorescence anisotropy which showed that none of these phospho-sugars can prevent the cooperative binding of CggR to its target DNA as FBP does (18). It is also noteworthy that the marginal peaks detected in the mass range corresponding to the 2:1 CggR/ O R complex are slightly more intense in the case of F6P than of GBP, which is also in good agreement with our previous findings indicating that F6P at high concentration (>5 mM) can indeed alter CggR/DNA-binding cooperativity (18). Unfortunately, the effect of the different sugars at high concentrations could not be tested here because of severe reduction in the signal-to-noise ratio of the mass spectra.…”

“…CggR is a member of the SorC family of bacterial transcriptional repressors, characterized by an N-terminal DNA-binding domain (DBD) followed by a phospho-sugar binding (PSB) domain homologous to glucosamine-6-phosphate deaminases from the NagB family (18). Other characterized members of this family are the sorbitol operon regulator SorC from Klebesiella pneumoniae (19) and the deoxyribonucleoside repressor DeoR from B. subtilis (20,21).…”

“…Other characterized members of this family are the sorbitol operon regulator SorC from Klebesiella pneumoniae (19) and the deoxyribonucleoside repressor DeoR from B. subtilis (20,21). Within this family, CggR and its paralogs in other Gram-positive bacteria define a subclass of repressors with a 100-residue-long DBD predicted to contain a MarR-type winged-helix (wH) motif (17,18). Recently, a few crystal structures from SorC family members have been deposited in the Protein Data Bank, notably several liganded forms of the PSB domain from B. subtilis CggR (22) and the full-length structure of K. pneumoniae SorC (23).…”

Crystal Structure of the Full-Length Sorbitol Operon Regulator SorC from Klebsiella pneumoniae: Structural Evidence for a Novel Transcriptional Regulation Mechanism

“…More recently, the binding activity and specificity of the CggR protein, the central glycolytic gene repressor, have been studied in detail. 4,5 Together with the DeoR protein of B. subtilis, these are the only members of the SorC family that have been purified and characterized in vitro. 6,7 K. pneumoniae SorC is the single transcriptional repressor of the sor operon, 8 which clusters seven genes (sorCDFBAME) that encode proteins involved in L-sorbose uptake and degradation.…”

Characterisation of Bacillus subtilis Transcriptional Regulators Involved in Metabolic Processes

“…A sequence analysis of CggR revealed a winged helix domain at the N-terminus, as found for example in transcription factors like SorC and DeoR [85]. On the contrary, the Cterminal domain, which comprises residues 100-343, showed similarities with glucose-6-phosphate deaminases, including NagB.…”