Purification and Characterization of Guanosine Diphospho-D-mannose Dehydrogenase

Crystal Structure of GDP-Mannose Dehydrogenase: A Key Enzyme of Alginate Biosynthesis in P. aeruginosa,

“…The crystal structure of UGD from Streptococcus pyogenes has been reported (10) and shows that the enzyme exists as a dimer. P. aeruginosa GMD (11) shows both allosteric and cooperative behavior, implying a higher order oligomeric state consistent with previous biochemical characterization of the enzyme (12). Here, we report determination of the crystal structure of P. aeruginosa † Supported by NIH Grant GM59653.…”

“…Many of the contacts are made by backbone atoms of the protein, which would tend to limit the accommodation of different orientations and types of bases. GMD is known to discriminate between substrates containing guanine and those with other bases (12).…”

“…For GMD, the opposite is not true since there appears to be room for glucose-based molecules to bind in the pocket, although the hydrogen bond to His217 would be unfulfilled. In fact, GDP-glucose has been reported to be an inhibitor of GMD but not a substrate (12). Site-directed mutagenesis can be used to confirm the apparent striking correlation between the structures and the binding specificities of these two enzymes.…”

“…Implications for Enzyme Mechanism. The reaction catalyzed by GMD is a 4-electron oxidation proposed to proceed via the pathway shown in Figure 1A (12). Two equivalents of NAD + are utilized to convert substrate to product in a bi uni uni bi ping-pong reaction mechanism.…”

“…On the basis of sequence alignments and comparison with the UGD structure, Cys268 of GMD was proposed to be the nucleophilic thiol ( , ). The importance of this residue is consistent with the sensitivity of the enzyme to thio-reactive reagents and a Cys268 to serine mutation that reduces enzyme activity by 95% ().…”