Structural Studies Of L-sorbose Dehydrogenase And Nucleotide-rhamnose Synthetase

It is well known thatKetogulonicignium vulgareY25could effectively oxidize L-sorbose to2-keto-L-gulonic acid (KGA), an industrial precursor of vitamin C. In this process, L-sorbose dehydrogenase is one of the key enzymes responsible for the production of2KGA. And quinoprotein(PQQ) was found to be indispensable for its activity as prostheticgroup.The crystal structure of the L-sorbose dehydrogenase (SDH) fromKetogulonicigenium vulgareY25has been determined at2.70A resolution using the molecular replacement method. The overall structure of SDH is similar to that of other quinoprotein dehydrogenases, consisting of an eight bladed β-propeller PQQ domain and protrusive loops. Each P-propeller folded into a W-like motif, consisting of four-strand antiparallel β-sheets, with the first β-strand (Q44-G50) participating in the last W8-motif. We identified a stable homodimer in crystals and demonstrated its existence in solution by sedimentation velocity measurement with Kd=167nM. The PQQ cofactor is coordinated to a calcium ion via its ring O5, N6and O7atoms, and the two components can be considered as a single entity. This entity is sandwiched between the side chains of Trp218below it and the side chains of Phe103above it of SDH protein, and interacts at the periphery of its three-rings system with SDH via hydrogen binding or metal coordination to nine amino acids side chains.By biochemical characterization of the SDH in vitro, using L-sorbose as substrate and cytochrome C551as electron acceptor.It is revealedthatcytochrome C551acting as physiological primary electron acceptor for SDH. And we proposed the possible respiratory metabolism pathway.In this paper, the other crystal structure of NRS/ER at2.70A resolution was solved, by use of the anomalous signal of zinc atoms (SAD) method. This enzyme is a biofunctional3,5-epimerase4-keto reductase for nucleotide-rhamnose synthesis in A. thaliana. Our work establishes the role of Zn ion in stabilizing the dimer interface. The overall topology of NRS/ER folds into a bean-shaped structure and the monomer can be subdivided into two domains of different function:cofactor-binding domain and substrate-binding domain. We also identified a stable homodimer in crystal and demonstrated its existence in solution by sedimentation velocity measurement, which is the character of SDR superfamily.To determine the cofactor and substrate specificity of NRS/ER, ITC was performed to investigate binding of NADH, NADPH, UDP and dTDP. The results showed that NRS/ER has a clear preference for NADPH to NADH, and alsofor dTDP to UDP.Site-directed mutation analysis suggests an essential role of Lys183in dTDP binding to NRS/ER.