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

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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.

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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.

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  1. Andrei M. Vacaru

    Present address: Department of Medicine, The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA

Authors and Affiliations

  1. Department of Enzymology, Institute of Biochemistry of the Romanian Academy, 296 Splaiul Independentei, 060031, Bucharest, Romania

    Georgiana Petrareanu, Mihaela C. Balasu, Andrei M. Vacaru, Aura E. Ionescu & Stefan E. Szedlacsek

  2. Department of Bioinformatics and Structural Biochemistry, Institute of Biochemistry of the Romanian Academy, 296 Splaiul Independentei, 060031, Bucharest, Romania

    Cristian V. A. Munteanu

  3. Department of Physical Chemistry, Faculty of Chemistry, University of Bucharest, Bd. Regina Elisabeta, No. 4-12, Bucharest, Romania

    Iulia Matei

  4. Department of Organic Chemistry, University POLITEHNICA, 1-7 Polizu Street, 011061, Bucharest, Romania

    Mihaela C. Balasu

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  1. Georgiana Petrareanu
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Georgiana Petrareanu and Mihaela C. Balasu contributed equally to this work.

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Petrareanu, G., Balasu, M.C., Vacaru, A.M. et al. Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics. Appl Microbiol Biotechnol 98, 7855–7867 (2014). https://doi.org/10.1007/s00253-014-5723-6

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