Part:BBa_K4584000

Corynebacter glutamicum LldR

LldR is a lactate binding transcription factor that regulates the transcription of the lldPRD operon (https://parts.igem.org/Part:BBa_K4584001). The proteins encoded (lactate permease and lactate dehydrogenase) within this operon are involved in metabolism of lactic acid in bacteria.

LldR is a lactate-responsive transcription factor belonging to the GntR family that governs the expression of the lldPRD operon in various bacterial species including E. coli and Corynebacterium glutamicum (Anzai et al.2023, Gao et Al. 2008). LldR represses the expression of the lldPRD operon by binding to operator region. In presence of L-lactate, the affinity of LldR to the operator region changes, resulting in downstream gene expression (Gao et al., 2008).

Members of the GntR family are characterised by a highly conserved winged helix-turn-helix motif (wHTH) in their N-terminus domain (NTD) responsible for DNA binding. This helix-turn-helix motif is comprised of a tri-helical core, with helices 2 and 3 interacting specifically with the major groove of the DNA. Following the helical core, two beta strands are linked through a small loop, referred to as the "wing" motif. However, these transcription factors display variations in their C-terminal effector-binding and oligomerization domains. (Jain, 2015) In the case of LldR, the C-terminal domain consists of seven helices that form a helical bundle responsible for binding to L-lactate and dimerization (Gao et al., 2008).

Figure 1 - (A) Overall structure of the LldR monomer with a DNA binding domain at the N terminal and a L -lactate binding domain at the C-terminal. (B) Close -up of the DNA binding domain highlighting the amino acids that interact with DNA.

The 2008 study by Gao et al. provided a structural characterization of LldR from Corynebacterium glutamicum. Through mutagenesis assays, they identified the residues that interact with DNA. Their findings highlighted the significance of Lys4, Glu31, Arg32, Arg42, Arg46, Glu47, Gly63, Gly65, and Arg67 in LldR for DNA binding. (Figure 3 and Figure 2B) Notably, with the exception of Arg67, all of these residues are either conserved or exhibit conservative changes in LldR and its homologues.

References: - Aguilera, L., Campos, E., Giménez, R., Badía, J., Aguilar, J., & Baldoma, L. (2008). Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. Journal of bacteriology, 190(8), 2997–3005. https://doi.org/10.1128/JB.02013-07 - Gao YG, Suzuki H, Itou H, Zhou Y, Tanaka Y, Wachi M, Watanabe N, Tanaka I, Yao M. Structural and functional characterization of the LldR from Corynebacterium glutamicum: a transcriptional repressor involved in L-lactate and sugar utilization. Nucleic Acids Res. 2008 Dec;36(22):7110-23. doi: 10.1093/nar/gkn827. Epub 2008 Nov 6. PMID: 18988622; PMCID: PMC2602784.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 187
  Illegal XhoI site found at 409
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 555
  
• 1000
  COMPATIBLE WITH RFC[1000]