Mechanistic and stereochemical studies of a unique dehydration catalyzed by CDP-4-keto-6-deoxy-D-glucose-3-dehydrase: a pyridoxamine 5'-phosphate dependent enzyme isolated from Yersinia pseudotuberculosis.
CDP-4-keto-6-deoxy-D-glucose-3-dehydrase (E1) purified from Yersinia pseudotuberculosis is a pyridoxamine 5'-phosphate (PMP) dependent enzyme which catalyzes the C-O bond cleavage at C-3 of a CDP-4-keto-6-deoxy-D-glucose substrate, a key step in the formation of 3,6-dideoxyhexoses. Since enzyme E1 utilizes the PMP cofactor in a unique manner, it is essential to establish its role in E1 catalysis. When an incubation was conducted in [18O]H2O, incorporation of 18O into positions C-3 and C-4 of the recovered substrate was observed. This result not only provided the evidence necessary to reveal the reversibility of E1 catalysis but also lent credence to the formation of a delta 3,4-glucoseen intermediate. In view of E1 catalysis being initiated by a C-4' deprotonation of the PMP-substrate complex, the stereochemical course of this step was examined using chemically synthesized (4'S)- and (4'R)-[4'-3H]PMP as probes. Our results clearly demonstrated that the stereochemistry of this deprotonation is pro-S specific, which is in agreement with the stereochemical consistency found with other vitamin B6 phosphate dependent enzymes. The fact that reprotonation at C-4' of the PMP-delta 3,4-glucoseen complex in the reverse direction of E1 catalysis was also found to be pro-S stereospecific strongly suggested that enzyme E1, like most of its counterparts, has the si face of its cofactor-substrate complex exposed to solvent and accessible to active-site catalytic groups as well.(ABSTRACT TRUNCATED AT 250 WORDS)
Weigel
T M
TM
Department of Chemistry, University of Minnesota, Minneapolis 55455.
Miller
V P
VP
Liu
H W
HW
D002384:Catalysis; D003837:Deoxy Sugars / Q000096:biosynthesis*; D006836:Hydro-Lyases / Q000378:metabolism*; D006868:Hydrolysis; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000378:metabolism; D013237:Stereoisomerism; D015011:Yersinia pseudotuberculosis / Q000201:enzymology*
D003837:Deoxy Sugars; D011733:Pyridoxamine; D010088:Oxidoreductases; C064052:CDP-6-deoxy-delta(3,4)-glucoseen reductase; C073141:CDP-4-keto-6-deoxy-D-glucose-3-dehydrase; D006836:Hydro-Lyases; C010627:pyridoxamine phosphate