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

Bioorganic Chemistry
Alejandro Yevenes, Perry A Frey

Abstract

The genes xpk1 and xpk2(Delta1-21) encoding phosphoketolase-1 and (Delta1-7)-truncated phosphoketolase-2 have been cloned from Lactobacillus plantarum and expressed in Escherichia coli. Both gene-products display phosphoketolase activity on fructose-6-phosphate in extracts. A N-terminal His-tag construct of xpk2(Delta1-21) was also expressed in E. coli and produced active His-tagged (Delta1-7)-truncated phosphoketolase-2 (hereafter phosphoketolase-2). Phosphoketolase-2 is activated by thiamine pyrophosphate (TPP) and the divalent metal ions Mg(2+), Mn(2+), or Ca(2+). Kinetic analysis and data from the literature indicate the activators are MgTPP, MnTPP, or CaTPP, and these species activate by an ordered equilibrium binding pathway, with Me(2+)TPP binding first and then fructose-6-phosphate. Phosphoketolase-2 accepts either fructose-6-phosphate or xylulose-5-phosphate as substrates, together with inorganic phosphate, to produce acetyl phosphate and either erythrose-4-phosphate or glyceraldehyde-3-phosphate, respectively. Steady state kinetic analysis of acetyl phosphate formation with either substrate indicates a ping pong kinetic mechanism. Product inhibition patterns with erythrose-4-phosphate indicate that an intermediate in ...Continue Reading

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Citations

Jul 8, 2010·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Georgiana PetrareanuStefan E Szedlacsek
Aug 10, 2010·Acta Crystallographica. Section F, Structural Biology and Crystallization Communications·Ryuichiro SuzukiKenji Yamamoto
Sep 12, 2015·Current Opinion in Biotechnology·Calvin Andrew HenardMichael Thomas Guarnieri
Jul 9, 2016·Oncotarget·Santiago Diaz-MoralliMarta Cascante

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