Ambient temperature structure of phosphoketolase from Bifidobacterium longum determined by serial femtosecond X-ray crystallography

Abstract

Phosphoketolase and transketolase are thiamine diphosphate-dependent enzymes and play a central role in the primary metabolism of bifidobacteria: the bifid shunt. The enzymes both catalyze phosphorolytic cleavage of xylulose 5-phosphate or fructose 6-phosphate in the first reaction step, but possess different substrate specificity in the second reaction step, where phosphoketolase and transketolase utilize inorganic phosphate (Pi) and D-ribose 5-phosphate, respectively, as the acceptor substrate. Structures of Bifidobacterium longum phosphoketolase holoenzyme and its complex with a putative inhibitor, phosphoenolpyruvate, were determined at 2.5 Å resolution by serial femtosecond crystallography using an X-ray free-electron laser. In the complex structure, phosphoenolpyruvate was present at the entrance to the active-site pocket and plugged the channel to thiamine diphosphate. The phosphate-group position of phosphoenolpyruvate coincided well with those of xylulose 5-phosphate and fructose 6-phosphate in the structures of their complexes with transketolase. The most striking structural change was observed in a loop consisting of Gln546-Asp547-His548-Asn549 (the QN-loop) at the entrance to the active-site pocket. Contrary to the conformation of the QN-loop that partially covers the entrance to the active-site pocket (`closed form') in the known crystal structures, including the phosphoketolase holoenzyme and its complexes with reaction intermediates, the QN-loop in the current ambient structures showed a more compact conformation with a widened entrance to the active-site pocket (`open form'). In the phosphoketolase reaction, the `open form' QN-loop may play a role in providing the binding site for xylulose 5-phosphate or fructose 6-phosphate in the first step, and the `closed form' QN-loop may help confer specificity for Pi in the second step.

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“…Over 20 enzymes (from prokaryotes and eukaryotes) dependent on thiamine pyrophosphate are recognized. Noteworthy examples include glyoxylate carboligase [ 47 ], transketolase [ 48 ], phosphoenolpyruvate decarboxylase [ 49 ], 2-hydroxyacyl-CoA lyase [ 50 ], pyruvate dehydrogenase complex [ 45 ], 2-hydroxyphytanoyl-CoA lyase [ 51 ], branched-chain amino acid enzyme [ 52 ], phosphoketolase [ 53 ], benzoyl formate decarboxylase, acetohydroxyacid synthase [ 54 ], pyruvate decarboxylase [ 55 ], 2-oxoglutarate dehydrogenase complex [ 56 ], sulfopyruvate decarboxylase [ 57 ], pyruvate ferredoxin oxidoreductase [ 58 ], and phenylpyruvate decarboxylase [ 59 ].…”

“…2-hydroxyacyl-CoA lyase [50], pyruvate dehydrogenase complex [45], 2-hydroxyphytanoyl-CoA lyase [51], branched-chain amino acid enzyme [52], phosphoketolase [53], benzoyl formate decarboxylase, acetohydroxyacid synthase [54], pyruvate decarboxylase [55], 2-oxoglutarate dehydrogenase complex [56], sulfopyruvate decarboxylase [57], pyruvate ferredoxin oxidoreductase [58], and phenylpyruvate decarboxylase [59].…”