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Applied Microbiology and Biotechnology

, Volume 98, Issue 18, pp 7855–7867 | Cite as

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

  • Georgiana Petrareanu
  • Mihaela C. Balasu
  • Andrei M. Vacaru
  • Cristian V. A. Munteanu
  • Aura E. Ionescu
  • Iulia Matei
  • Stefan E. SzedlacsekEmail author
Biotechnologically relevant enzymes and proteins

Abstract

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 K m values for X5P and F6P suggesting the highest capacity for substrate binding. PK-Ll has the largest k cat 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.

Keywords

Circular dichroism Expression and purification Kinetic parameters Mass spectrometry Phosphoketolase 

Notes

Acknowledgement

We are deeply indebted to Octavian Bârzu for the initial research idea of the research project behind this manuscript. We are grateful to Alexei Sorokin (Institut National de la Recherche Agronomique, Jouy en Josas, France) for the L. lactis genomic fragment containing the PK-Ll gene, to Medana Zamfir for the L. mesenteroides strain (Institute of Biology of the Romanian Academy, Bucharest, Romania) and to Octavian Popescu (University Babes-Bolyai, Cluj Napoca, Romania) for the pET28b-PK vector containing the PK-Pa gene. We thank Mihaela Mentel for her kind help during manuscript editing. G.P., M.B., A.V and S.S. work was supported by grants from BIOTECH, CNMP, CNCSIS, European Social Fund, Alexander von Humboldt Foundation, Romanian National Authority for Scientific Research CNCS–UEFISCDI, projects 02-2-PED-427, CEEX 1/2005, PN-II-ID-PCE-210/2007, POSDRU/89/1.5/S/60746, PN-II-PT-PCCA- 79/2012 and by the Romanian Academy project no. 5 of the Institute of Biochemistry of the Romanian Academy. C.V.A.M. acknowledges support from Romanian National Authority for Scientific Research CNCS–UEFISCDI, project code PN-II-ID-PCE-2011-3-0342 no. 181/2011 for mass spectrometry work. Circular dichroism determinations were supported by a grant of the Romanian National Authority for Scientific Research, CNCS–UEFISCDI, project PN-II-RU-TE-2011-3-0281.

Supplementary material

253_2014_5723_MOESM1_ESM.pdf (50 kb)
ESM 1 (PDF 50 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Georgiana Petrareanu
    • 1
  • Mihaela C. Balasu
    • 1
    • 4
  • Andrei M. Vacaru
    • 1
    • 5
  • Cristian V. A. Munteanu
    • 2
  • Aura E. Ionescu
    • 1
  • Iulia Matei
    • 3
  • Stefan E. Szedlacsek
    • 1
    Email author
  1. 1.Department of EnzymologyInstitute of Biochemistry of the Romanian AcademyBucharestRomania
  2. 2.Department of Bioinformatics and Structural BiochemistryInstitute of Biochemistry of the Romanian AcademyBucharestRomania
  3. 3.Department of Physical Chemistry, Faculty of ChemistryUniversity of BucharestBucharestRomania
  4. 4.Department of Organic ChemistryUniversity POLITEHNICABucharestRomania
  5. 5.Department of MedicineThe Tisch Cancer Institute, Icahn School of Medicine at Mount SinaiNew YorkUSA

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