Cloning, expression, purification, cofactor requirements, and steady state kinetics of phosphoketolase-2 from Lactobacillus plantarum

Yévenes, Alejandro; Frey, Perry A.

doi:10.1016/j.bioorg.2008.03.002

Cited by 29 publications

(54 citation statements)

References 23 publications

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“…Dehydration Mechanism-Observation of the DHEThDP and AcThDP adducts after soaking the crystals in F6P for very short and relatively long periods of time, respectively, supports the previous notion that the dehydration occurs in the absence of P i (22,33). The water molecule observed near AcThDP is held by four hydrogen bonds with the side chain N atoms of His-97 and His-142 and the main chain O atoms of Gly-154 and Gly-155 (Fig.…”

Section: (C)supporting

confidence: 82%

“…Based on these observations, we concluded that the covalent adduct is an acetyl group. It has been proposed that AcThDP formed after dehydration is a stable intermediate of PK in the absence of the acceptor, P i (22,33). One of the two terminal atoms is located close to the NE2 atom of His-553 (2.5 Å) and the N4Ј atom of the pyrimidine ring (3.2 Å) (Fig.…”

Section: (C)mentioning

confidence: 99%

“…2) (22). However, the subsequent reaction catalyzed by PK is distinct from TK at the following two points; 1) the dehydration reaction occurs, and 2) the acceptor substrate P i is believed to attack the possible 2-acetyl-ThDP (AcThDP) intermediate, similar to the case of acetyl-P-producing POX.…”

mentioning

confidence: 99%

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Crystal Structures of Phosphoketolase

Suzuki

Katayama

Kim

et al. 2010

Journal of Biological Chemistry

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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.

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Section: (C)supporting

confidence: 82%

Section: (C)mentioning

confidence: 99%

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Crystal Structures of Phosphoketolase

Suzuki

Katayama

Kim

et al. 2010

Journal of Biological Chemistry

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“…Acetyl-CoA ϩ P i ↔ acetyl phosphate ϩ CoA (4) Xfp has been biochemically and kinetically characterized from several bacterial species, including Lactobacillus plantarum (referred to by Yevenes and Frey as L. plantarum Xpk2) (2), Bifidobacterium spp. (1,4), Lactococcus lactis (5), Leuconostoc mesenteroides (5), and Pseudomonas aeruginosa (5), and, more recently, one fungal species, Cryptococcus neoformans Xfp2 (6).…”

mentioning

confidence: 99%

“…(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).…”

mentioning

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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Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics

Petrareanu

Balasu

Vacaru

et al. 2014

Appl Microbiol Biotechnol

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Phosphoketolases (PKs) are large thiamine pyrophosphate (TPP)-dependent enzymes playing key roles in a number of essential pathways of carbohydrate metabolism. The putative PK genes of Lactococcus lactis (Ll) and Leuconostoc mesenteroides (Lm) were cloned in a prokaryotic vector, and the encoded proteins were expressed and purified yielding high purity proteins termed PK-Ll and PK-Lm, respectively. Similarly, the PK gene of Pseudomonas aeruginosa was expressed, and the corresponding protein (PK-Pa) was purified to homogeneity. The amino acid sequences predicted on the basis of genes' nucleotide sequences were confirmed by mass spectrometry and display low relative similarities. Circular dichroism (CD) spectra of these proteins predict higher α-helix than β-strand contents. In addition, it is predicted that PK-Ll contains tightly packed domains. Enzymatic analysis showed that all three recombinant proteins, despite their dissimilar amino acid sequences, are active PKs and accept both xylulose 5-phosphate (X5P) and fructose 6-phosphate (F6P) as substrates. However, they display substantially higher preference for X5P than for F6P. Kinetic measurements indicated that PK-Pa has the lowest Km values for X5P and F6P suggesting the highest capacity for substrate binding. PK-Ll has the largest kcat values for both substrates. Nevertheless, in terms of substrate specificity constant, PK-Pa has been found to be the most active PK against X5P. Structural models for all three analysed PKs predict similar folds in spite of amino acid sequence dissimilarities and contribute to understanding the enzymatic peculiarities of PK-Pa compared to PK-Ll and PK-Lm.

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Cloning, expression, purification, cofactor requirements, and steady state kinetics of phosphoketolase-2 from Lactobacillus plantarum

Cited by 29 publications

References 23 publications

Crystal Structures of Phosphoketolase

Crystal Structures of Phosphoketolase

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

Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics

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