Metabolic pathways have traditionally been described in terms of biochemical reactions and metabolites. Using structural genomics and systems biology, we generated a three-dimensional reconstruction of the central metabolic network of the bacterium, Thermotoga maritima (TM). The network encompassed 478 proteins of which 120 were determined by experiment and 358 were modeled. Structural analysis revealed that proteins forming the network are dominated by a small number (only 182) of basic shapes (folds) performing diverse, but mostly related functions. Most of these folds are already present in the essential core (~30%) of the network, and its expansion by nonessential proteins is achieved with relatively few additional folds. Thus, integration of structural data with networks analysis generates insight into the function, mechanism and evolution of biological networks.

PDB Reference: YkfC-L-Ala--D-Glu complex, 3h41.Dipeptidyl-peptidase VI from Bacillus sphaericus and YkfC from Bacillus subtilis have both previously been characterized as highly specific -d-glutamyll-diamino acid endopeptidases. The crystal structure of a YkfC ortholog from Bacillus cereus (BcYkfC) at 1.8 Å resolution revealed that it contains two N-terminal bacterial SH3 (SH3b) domains in addition to the C-terminal catalytic NlpC/P60 domain that is ubiquitous in the very large family of cell-wall-related cysteine peptidases. A bound reaction product (l-Ala--d-Glu) enabled the identification of conserved sequence and structural signatures for recognition of l-Ala and -d-Glu and, therefore, provides a clear framework for understanding the substrate specificity observed in dipeptidyl-peptidase VI, YkfC and other NlpC/P60 domains in general. The first SH3b domain plays an important role in defining substrate specificity by contributing to the formation of the active site, such that only murein peptides with a free N-terminal alanine are allowed. A conserved tyrosine in the SH3b domain of the YkfC subfamily is correlated with the presence of a conserved acidic residue in the NlpC/P60 domain and both residues interact with the free amine group of the alanine. This structural feature allows the definition of a subfamily of NlpC/P60 enzymes with the same N-terminal substrate requirements, including a previously characterized cyanobacterial l-alanine--d-glutamate endopeptidase that contains the two key components (an NlpC/P60 domain attached to an SH3b domain) for assembly of a YkfC-like active site.

Crystal structures of two homologous peptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme at 1.05 Å and 1.60 Å resolution represent the first structures of a large class of cell-wall, cysteine peptidases that contain an N-terminal bacterial SH3-like domain (SH3b) and a C-terminal NlpC/P60 cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that that these two proteins act as γ-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.