Biosynthesis of Pyridoxal Phosphate STEPS 3-4 OF FIGURE 25.1:TRANSAMINATION AND OXIDATION/DECARBOXYLATION 3-Hydroxy-4-phosphohydroxy-2-ketobutyrate undergoes transamination on reaction with a-ketoglutarate Occurs by usual PLP-dependent mechanism Product 4-phosphohydroxythreonine is oxidized by NAD+to give intermediate B-keto ester which undergoes concurrent decarboxylation and yields 1-amino-3-hydroxyacetone 3- phosphate
STEPS 3-4 OF FIGURE 25.1: TRANSAMINATION AND OXIDATION/DECARBOXYLATION 3-Hydroxy-4-phosphohydroxy-2-ketobutyrate undergoes transamination on reaction with a-ketoglutarate ▪ Occurs by usual PLP-dependent mechanism Product 4-phosphohydroxythreonine is oxidized by NAD+ to give intermediate b-keto ester which undergoes concurrent decarboxylation and yields 1-amino-3-hydroxyacetone 3- phosphate Biosynthesis of Pyridoxal Phosphate
Biosynthesis of Pyridoxal Phosphate Decarboxylation of b-keto ester yields 1-amino-3- hydroxyacetone 3-phosphate 0 a-Ketoglutarate NAD+ Glutamate NADH/H+ C=0 H3N H H OH H OH CH2OPO32- CH2OPO32- 3-Hydroxy-4-phospho- 4-Phosphohydroxy- hydroxy-2-ketobutyrate threonine c02 H3N H A H3N 0P032 C=0 1-Amino-3-hydroxy- CH20P032 acetone 3-phosphate A B-keto ester
▪ Decarboxylation of b-keto ester yields 1-amino-3- hydroxyacetone 3-phosphate Biosynthesis of Pyridoxal Phosphate
Biosynthesis of Pyridoxal Phosphate STEP 5 OF FIGURE 25.1:FORMATION OF 1- DEOXYXYLULOSE 5-PHOSPHATE 1-Amino-3-hydroxyacetone 3-phosphate from step 4 reacts in step 6 with 1-deoxyxylulose 5-phosphate (DXP) DXP arises in step 5 by an aldol-like condensation of D- glyceraldehyde 3-phosphate with pyruvate Reaction is thiamin-dependent Catalyzed by DXP synthase Hydroxyethylthiamin (HETPP)adds to glyceraldehyde 3- phosphate in an aldol-like reaction Tetrahedral intermediate expels TPP ylide as a leaving group yielding DXP
STEP 5 OF FIGURE 25.1: FORMATION OF 1- DEOXYXYLULOSE 5-PHOSPHATE 1-Amino-3-hydroxyacetone 3-phosphate from step 4 reacts in step 6 with 1-deoxyxylulose 5-phosphate (DXP) DXP arises in step 5 by an aldol-like condensation of Dglyceraldehyde 3-phosphate with pyruvate ▪ Reaction is thiamin-dependent ▪ Catalyzed by DXP synthase ▪ Hydroxyethylthiamin (HETPP) adds to glyceraldehyde 3- phosphate in an aldol-like reaction ▪ Tetrahedral intermediate expels TPP ylide as a leaving group yielding DXP Biosynthesis of Pyridoxal Phosphate
Biosynthesis of Pyridoxal Phosphate Figure 25.4 Mechanism of step Thiamin diphosphate ylide adds to the 5 in pyridoxal phosphate biosynthesis,the The addition product containsa C-N o a keto acid.It th erefore loses CO thiamin-dependent aiving the enamine HE TPP aldol reaction of D- glyceraldehyde 3- phosphate with pyruvate to give 1- deoxyxylulose 5- phosphate Cleavage of the adduct in a retro-aldol H OH
Figure 25.4 Mechanism of step 5 in pyridoxal phosphate biosynthesis, the thiamin-dependent aldol reaction of Dglyceraldehyde 3- phosphate with pyruvate to give 1- deoxyxylulose 5- phosphate Biosynthesis of Pyridoxal Phosphate
Biosynthesis of Pyridoxal Phosphate STEP 6 OF FIGURE 25.1:CONDENSATION AND CYCLIZATION 1-Deoxy-D-xylulose 5-phosphate is dephosphorylated and then condenses with 1-amino-3-hydroxyacetone 3- phosphate to give pyridoxine 5'-phosphate Reaction begins with formation of enamine followed by loss of water to form an enol that contains a ketone group six atoms removed The enol adds to the ketone in an intramolecular aldol reaction to form a six-membered ring which then loses water
STEP 6 OF FIGURE 25.1: CONDENSATION AND CYCLIZATION 1-Deoxy-D-xylulose 5-phosphate is dephosphorylated and then condenses with 1-amino-3-hydroxyacetone 3- phosphate to give pyridoxine 5′-phosphate ▪ Reaction begins with formation of enamine followed by loss of water to form an enol that contains a ketone group six atoms removed ▪ The enol adds to the ketone in an intramolecular aldol reaction to form a six-membered ring which then loses water Biosynthesis of Pyridoxal Phosphate