Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics
- 433 Downloads
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.
KeywordsCircular dichroism Expression and purification Kinetic parameters Mass spectrometry Phosphoketolase
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.
- Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, Dufayard JF, Guindon S, Lefort V, Lescot M, Claverie JM, Gascuel O (2008) Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36(Web Server issue):W465–W469. doi: 10.1093/nar/gkn180 PubMedCentralPubMedCrossRefGoogle Scholar
- Fleige C, Kroll J, Steinbuchel A (2011) Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16. Appl Microbiol Biotechnol 91(3):769–776. doi: 10.1007/s00253-011-3284-5
- Imperi F, Ciccosanti F, Perdomo AB, Tiburzi F, Mancone C, Alonzi T, Ascenzi P, Piacentini M, Visca P, Fimia GM (2009) Analysis of the periplasmic proteome of Pseudomonas aeruginosa, a metabolically versatile opportunistic pathogen. Proteomics 9(7):1901–1915. doi: 10.1002/pmic.200800618 PubMedCrossRefGoogle Scholar
- Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A, Pavlova N, Karamychev V, Polouchine N, Shakhova V, Grigoriev I, Lou Y, Rohksar D, Lucas S, Huang K, Goodstein DM, Hawkins T, Plengvidhya V, Welker D, Hughes J, Goh Y, Benson A, Baldwin K, Lee JH, Diaz-Muniz I, Dosti B, Smeianov V, Wechter W, Barabote R, Lorca G, Altermann E, Barrangou R, Ganesan B, Xie Y, Rawsthorne H, Tamir D, Parker C, Breidt F, Broadbent J, Hutkins R, O’Sullivan D, Steele J, Unlu G, Saier M, Klaenhammer T, Richardson P, Kozyavkin S, Weimer B, Mills D (2006) Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci U S A 103(42):15611–15616. doi: 10.1073/pnas.0607117103 PubMedCentralPubMedCrossRefGoogle Scholar
- Nagy CI, Lupan I, Ferencz BK, Popescu O (2007) Cloning and expression of the gene encoding phosphoketolase in Pseudomonas aeruginosa 15442. Ann West Univ Timisoara Ser Chem 16(3):73–80Google Scholar
- Panagiotou G, Andersen MR, Grotkjaer T, Regueira TB, Hofmann G, Nielsen J, Olsson L (2008) Systems analysis unfolds the relationship between the phosphoketolase pathway and growth in Aspergillus nidulans. PLoS One 3(12):e3847. doi: 10.1371/journal.pone.0003847 PubMedCentralPubMedCrossRefGoogle Scholar
- Petrareanu G, Balasu MC, Zander U, Scheidig AJ, Szedlacsek SE (2010) Preliminary X-ray crystallographic analysis of the D-xylulose 5-phosphate phosphoketolase from Lactococcus lactis. Acta Crystallogr F 66(7):805–807Google Scholar
- Posthuma CC, Bader R, Engelmann R, Postma PW, Hengstenberg W, Pouwels PH (2002) Expression of the xylulose 5-phosphate phosphoketolase gene, xpkA, from Lactobacillus pentosus MD363 is induced by sugars that are fermented via the phosphoketolase pathway and is repressed by glucose mediated by CcpA and the mannose phosphoenolpyruvate phosphotransferase system. Appl Environ Microbiol 68(2):831–837PubMedCentralPubMedCrossRefGoogle Scholar
- Tanaka K, Komiyama A, Sonomoto K, Ishizaki A, Hall SJ, Stanbury PF (2002) Two different pathways for D-xylose metabolism and the effect of xylose concentration on the yield coefficient of L-lactate in mixed-acid fermentation by the lactic acid bacterium Lactococcus lactis IO-1. Appl Microbiol Biotechnol 60(1–2):160–167. doi: 10.1007/s00253-002-1078-5 PubMedGoogle Scholar
- Whitmore L, Wallace BA (2009) Advances in biomedical spectroscopy—modern techniques for circular dichroism and synchrotron radiation circular dichroism spectroscopy, vol 1. IOS Press, AmsterdamGoogle Scholar
- Yuan J, Zhu L, Liu X, Li T, Zhang Y, Ying T, Wang B, Wang J, Dong H, Feng E, Li Q, Wang J, Wang H, Wei K, Zhang X, Huang C, Huang P, Huang L, Zeng M, Wang H (2006) A proteome reference map and proteomic analysis of Bifidobacterium longum NCC2705. Mol Cell Proteomics 5(6):1105–1118. doi: 10.1074/mcp.M500410-MCP200 PubMedCrossRefGoogle Scholar