title: Phosphoketolases from Lactococcus lactis, Leuconostoc mesenteroides and Pseudomonas aeruginosa: dissimilar sequences, similar substrates but distinct enzymatic characteristics.
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 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.
authors.affiliation: Department of Enzymology, Institute of Biochemistry of the Romanian Academy, 296 Splaiul Independentei, 060031, Bucharest, Romania, georgianapetrareanu@yahoo.com.
authors.forename: Georgiana
authors.initials: G
authors.lastname: Petrareanu
authors.forename: Mihaela C
authors.initials: MC
authors.lastname: Balasu
authors.forename: Andrei M
authors.initials: AM
authors.lastname: Vacaru
authors.forename: Cristian V A
authors.initials: CV
authors.lastname: Munteanu
authors.forename: Aura E
authors.initials: AE
authors.lastname: Ionescu
authors.forename: Iulia
authors.initials: I
authors.lastname: Matei
authors.forename: Stefan E
authors.initials: SE
authors.lastname: Szedlacsek
mesh_terms: D000446:Aldehyde-Lyases; D007700:Kinetics; D013294:Lactococcus lactis / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism*; D007934:Leuconostoc / Q000201:enzymology*; D013058:Mass Spectrometry; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D000446:Aldehyde-Lyases; C022367:phosphoketolase