Crystal structure of <i>Bifidobacterium Longum</i> phosphoketolase; key enzyme for glucose metabolism in <i>Bifidobacterium</i>

Takahashi, Kazutoshi; Tagami, Uno; Shimba, Nobuhisa; Kashiwagi, Tatsuki; Ishikawa, K.; Suzuki, Eiichiro

doi:10.1016/j.febslet.2010.07.043

Cited by 21 publications

(35 citation statements)

References 30 publications

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“…Therefore, the product E4P is formed by the interaction of TPP and F6P in the absence of the second substrate, P i . The crystal structure of Bifidobacterium longum Xfp confirmed the reaction mechanism proposed by Yevenes and Frey (19). We suspect that C. neoformans Xfp2 follows the same reaction mechanism as the bacterial Xfps but with the added complexity of allosteric regulation that influences substrate binding affinity.…”

Section: Figsupporting

confidence: 80%

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

2014

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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.

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Section: Figsupporting

confidence: 80%

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

2014

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“…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).…”

Section: Resultsmentioning

confidence: 99%

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

Glenn

Smith

2015

J. Bacteriol.

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Xylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), which catalyzes the conversion of xylulose 5-phosphate (X5P) or fructose 6-phosphate (F6P) to acetyl phosphate, plays a key role in carbohydrate metabolism in a number of bacteria. Recently, we demonstrated that the fungal Cryptococcus neoformans Xfp2 exhibits both substrate cooperativity for all substrates (X5P, F6P, and P i ) and allosteric regulation in the forms of inhibition by phosphoenolpyruvate (PEP), oxaloacetic acid (OAA), and ATP and activation by AMP (K. Glenn, C. Ingram-Smith, and K. S. Smith. Eukaryot Cell 13:657-663, 2014). Allosteric regulation has not been reported previously for the characterized bacterial Xfps. Here, we report the discovery of substrate cooperativity and allosteric regulation among bacterial Xfps, specifically the Lactobacillus plantarum Xfp. L. plantarum Xfp is an allosteric enzyme inhibited by PEP, OAA, and glyoxylate but unaffected by the presence of ATP or AMP. Glyoxylate is an additional inhibitor to those previously reported for C. neoformans Xfp2. As with C. neoformans Xfp2, PEP and OAA share the same or possess overlapping sites on L. plantarum Xfp. Glyoxylate, which had the lowest half-maximal inhibitory concentration of the three inhibitors, binds at a separate site. This study demonstrates that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfp enzymes, yet important differences exist between the enzymes in these two domains. IMPORTANCEXylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp) plays a key role in carbohydrate metabolism in a number of bacteria. Although we recently demonstrated that the fungal Cryptococcus Xfp is subject to substrate cooperativity and allosteric regulation, neither phenomenon has been reported for a bacterial Xfp. Here, we report that the Lactobacillus plantarum Xfp displays substrate cooperativity and is allosterically inhibited by phosphoenolpyruvate and oxaloacetate, as is the case for Cryptococcus Xfp. The bacterial enzyme is unaffected by the presence of AMP or ATP, which act as a potent activator and inhibitor of the fungal Xfp, respectively. Our results demonstrate that substrate cooperativity and allosteric regulation may be common properties among bacterial and eukaryotic Xfps, yet important differences exist between the enzymes in these two domains. Xylulose 5-phosphate (X5P)/fructose 6-phosphate (F6P) phosphoketolase (Xfp), a member of the thiamine pyrophosphate (TPP)-dependent enzyme family, catalyzes the production of acetyl phosphate from the breakdown of xylulose 5-phosphate (equation 1; EC 4.1.2.9) or fructose 6-phosphate (equation 2; EC 4.1.2.22). In lactic acid bacteria and bifidobacteria, Xfp partners with either acetate kinase (Ack) to generate acetate and ATP (equation 3) or phosphotransacetylase (Pta) to generate acetyl coenzyme A (acetyl-CoA) and P i (equation 4) (1, 2). More recently, Xfp open reading frames (ORFs) have been discovered in euascomycete and basidiomycete fungi...

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“…The structure and mechanism of transketolase have been extensively studied, 121) but the three-dimensional structure and reaction mechanism of phosphoketolase has long been enigmatic, although phosphoketolase was discovered more than 50 years ago. [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.…”

Section: Bifid Shuntmentioning

confidence: 99%

Unique Sugar Metabolic Pathways of Bifidobacteria

Fushinobu

2010

Bioscience, Biotechnology, and Biochemistry

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Crystal structure of Bifidobacterium Longum phosphoketolase; key enzyme for glucose metabolism in Bifidobacterium

Cited by 21 publications

References 30 publications

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

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

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

Unique Sugar Metabolic Pathways of Bifidobacteria

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