Crystal Structures of Phosphoketolase

Abstract

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H 2 O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (␣,␤-dihydroxyethyl ThDP and 2-acetylThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

Allosteric Regulation of Lactobacillus plantarum Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase (Xfp)

“…We have recently shown that C. neoformans Xfp2 displays substrate cooperativity and is subject to allosteric regulation (6), neither of which has been reported for any of the bacterial Xfp enzymes (1,4,5,11), including the L. plantarum Xfp (2). To establish whether substrate cooperativity exists for L. plantarum Xfp, apparent kinetic parameters (Table 1) were determined in the acetyl phosphate-forming direction for the substrates F6P and P i by fitting experimental data to the Hill equation (equation 5), in which a Hill constant (h) greater than 1.0 represents positive cooperativity and a Hill constant less than 1.0 represents negative cooperativity (12,13).…”

“…(1,4), Lactococcus lactis (5), Leuconostoc mesenteroides (5), and Pseudomonas aeruginosa (5), and, more recently, one fungal species, Cryptococcus neoformans Xfp2 (6). The Bifidobacterium Xfp and the L. plantarum, L. lactis, L. mesenteroides, and P. aeruginosa Xfps displayed dual substrate specificity for both substrates X5P and F6P and followed Michaelis-Menten kinetics (1,2,4,5). C. neoformans Xfp2 also displays dual substrate specificity but does not follow Michaelis-Menten kinetics (6).…”

Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

“…In determining the kinetic parameters in the acetyl phosphate-forming direction, plots of substrate concentration versus velocity were found to be sigmoidal rather than hyperbolic, as would be expected for enzymes that follow Michaelis-Menten kinetics. This is the first demonstration of the existence of substrate cooperative binding among Xfp enzymes; this was not reported in previous characterizations of bacterial Xfps (7,9,10). Apparent kinetic parameters (Table 1) were determined by fitting experimental data to the Hill equation (see above), with a Hill constant greater than 1.0 representing positive cooperativity and a Hill constant less than 1.0 representing negative cooperativity (15,16).…”

“…and Lactobacillus plantarum have been purified and kinetically characterized (7,9). The characterized bacterial Xfp enzymes show dual substrate specificity with X5P and F6P and follow Michaelis-Menten kinetics (7,9,10). Here we report the first biochemical and kinetic characterization of eukaryotic Xfp, the C. neoformans Xfp2.…”

Unique Sugar Metabolic Pathways of Bifidobacteria

“…[122][123][124] Very recently, crystallization of phosphoketolase from Lactococcus lactis 125) and structure determination of a substrate-free form of XFPK from B. longum JCM1217 126) were reported. Our group also succeeded in crystallization 127) and structure determination 128) of XFPK from B. breve (Fig. 6B).…”