Structural and functional analysis of Vitamin K2 synthesis protein MenD.
Here we describe in detail the crystal structures of the Vitamin K(2) synthesis protein MenD, from Escherichia coli, in complex with thiamine diphosphate (ThDP) and oxoglutarate, and the effects of cofactor and substrate on its structural stability. This is the first reported structure of MenD in complex with oxoglutarate. The residues Gly472 to Phe488 of the active site region are either disordered, or in an open conformation in the MenD oxoglutarate complex structure, but adopt a closed conformation in the MenD ThDP complex structure. Biospecific-interaction analysis using surface plasmon resonance (SPR) technology reveals an affinity for ThDP and oxoglutarate in the nanomolar range. Biochemical and structural analysis confirmed that MenD is highly dependent on ThDP for its structural stability. Our structural results combined with the biochemical assay reveal novel features of the enzyme that could be utilized in a program of rational structure-based drug design, as well as in helping to enhance our knowledge of the menaquinone synthesis pathway in greater detail.
Priyadarshi
Amit
A
Biomedical Research Center, Korea Institute of Science and Technology, Seoul 136-791, South Korea.
Kim
Eunice Eunkyeong
EE
Hwang
Kwang Yeon
KY
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011771:Pyruvate Oxidase / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013835:Thiamine Pyrophosphate / Q000737:chemistry / Q000378:metabolism; D024482:Vitamin K 2 / Q000378:metabolism*
D029968:Escherichia coli Proteins; D024482:Vitamin K 2; C534108:2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase, E coli; D011771:Pyruvate Oxidase; D013835:Thiamine Pyrophosphate