Cofactors and Coenzymes bound to Structural Genomics Targets

Cofactors and Coenzymes bound to Structural Genomics Targets 

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on solving the structures of potential drug targets in pathogenic organisms. These proteins are highly varied in nature and essential to the pathogenic organism. Many of these targets utilize coenzymes or cofactors such as pyridoxal phosphate (PLP), flavin mononucleotide (FMN) or nicotinamide adenine dinucleotide (NAD). In approximately 15% of our solved structures, a coenzyme or cofactor bound to the target protein during expression in E. coli, co-purified along with the target protein, and was observed in the crystal structure. In most cases the identity of the small molecule is readily apparent based on homologous structures and the experimental electron density. Here we present two examples of a cofactor and a coenzyme bound to the potential drug target. In other cases, the identity of the small molecule can not be determined based on the electron density (e.g. PDB ID 3LB5).

FMN bound to N-ethylmaleimidine reductase from Burkholderia pseudomallei 

We solved a 2.3 Å resolution crystal structure of N-ethylmaleimidine reductase from Burkholderia pseudomallei, the causative reagent of melioidosis (targetDB entry BupsA.00093.a; PDB ID 3GKA; Figure 1). The data contained excellent omit electron density for the cofactor FMN, the phosphorylated analog of riboflavin (vitamin B2) which is used as a cofactor by oxidoreductases (Figure 2).

Figure 1. Crystal structure of FMN bound to N-ethylmaleimidine reductase from Burkholderia pseudomallei. The 2.3 Å resolution crystal structure is shown in ribbons representation with a molecular surface rendering. The cofactor FMN is bound in the active site with omit electron density (fo-fc) shown in green meshcontoured at 4.0 s.

Figure 2. Evidence for FMN binding to N-ethylmaleimidine reductase from Burkholderia pseudomallei. Omit electron density (fo-fc) at left shown in green mesh contoured at 4.0 s and refined electron density (2fo-fc) at right shown in blue mesh contoured at 1.0 s .

PLP bound to alanine racemase from Bartonella henselae 

In another example, we solved a 2.05 Å resolution crystal structure of alanine racemase from Bartonella henselae, a tick-born pathogen that causes cat-scratch disease (targetDB entry BaheA.00339.a; PDB ID 3KW3). The quaternary structure of B. henselae alanine racemase is dimeric, featuring one solvent exposed active site and one closed active site (Figure 3). Homologous structures in the PDB had low sequence identity and high gap percentage relative to BaheA.00339.a and resulted in poor molecular replacement solutions. Therefore, we solved a 2.95 Å resolution structure using combined iodide ion    SAD/molecular replacement in Phaser Experimental Phases (Phaser EP) prior to refinement of the high resolution native data set (see previous Featured Story on the use of iodide ions for SAD phasing). Based on the omit electron density pyridoxal phosphate (PLP) was present in the structure (Figure 4). Furthermore, the electron density was convincing that PLP had formed a covalent bond via a Schiff base with Lysine 47 of the protein. This covalent interaction is often observed for enzymes which use PLP as a coenzyme, and is denoted LLP in the coordinate file.



Figure 3. Dimeric structure of alanine racemase from Bartonella henselae. The crystal structure (PDB ID 3KW3) is shown as a ribbons diagram (left) and molecular surface representation (right). The coenzyme pyridoxal phosphate (PLP) is covalently bound to a lysine residue and shown in sticks representation with omit electron density (fo-fc) shown in green mesh contoured at 3.0 s. The active site of the left monomer features a solvent exposed active site, whereas the active site of the monomer on the right is solvent inaccessible




Figure 4. Coenzyme PLP covalently bound to a lysine residue in alanine racemase from Bartonella henselae. Omit electron density (fo-fc) at left shown in green mesh contoured at 4.0 s and refined electron density (2fo-fc) at right shown in blue mesh contoured at 1.0 s.