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

Abstract

Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogen Cryptococcus neoformans, which has two XFP genes (designated XFP1 and XFP2). Our kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics. C. neoformans Xfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC 50 ) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 M AMP is capable of increasing Xfp2 activity by 24.8% ؎ 1.0% (mean ؎ standard error of the mean), while 50 M prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogen Cryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.

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

“…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). L. plantarum Xfp displays negative cooperativity for P i , as indicated by a Hill constant of 0.68 Ϯ 0.02 (Table 1), similar to the Hill constant of 0.59 Ϯ 0.03 for the C. neoformans enzyme (6). The K 0.5 value of 1.0 Ϯ 0.1 mM for P i (K 0.5 is a kinetic parameter for enzymes that display substrate cooperativity and is similar to K m in defining the substrate concentration required to obtain half-maximum activity) is similar to the K m previously determined for the L. plantarum enzyme (2).…”

“…We recently determined that a fungal Xfp, C. neoformans Xfp2, displayed both substrate cooperativity (positive cooperativity for F6P and negative cooperativity for P i ) and allosteric regulation in the form of inhibition through the binding of ATP, PEP, and OAA and activation by the binding of AMP (6). Here, we report the discovery of substrate cooperativity and allosteric regulation for bacterial L. plantarum Xfp.…”

“…In regard to C. neoformans Xfp2 and L. plantarum Xfp regulation by excess PEP and OAA, the presence of these intermediates indicate the energy needs of the cell have been met. The cell can switch from glycolysis, the breakdown of glucose, to gluconeogenesis to synthesize and ultimately store glucose until it is needed; therefore, Xfp may be inhibited by these intermediates in order to limit additional energy production through the Xfp/Ack pathway (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). Instead, kinetic characterization of C. neoformans Xfp2 indicated the existence of both substrate cooperativity and allosteric regulation.…”

“…(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).…”