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Biochemistry |
Unité de Physiologie Microbienne, Département de Biochimie et Génétique Moléculaire, Institut Pasteur (CNRS, URA 1129), 28 rue du Docteur Roux, 75724 Paris, France1
Institut für Mikrobiologie, Fachrichtung 13.3, Universität des Saarlandes, D-66041 Saarbrücken, Germany2
Author for correspondence: Nicole Tandeau de Marsac. Tel: +33 1 45 68 8415. Fax: +33 1 40 61 3042. e-mail: ntmarsac{at}pasteur.fr
The urease from the picoplanktonic oceanic Prochlorococcus marinus sp. strain PCC 9511 was purified 900-fold to a specific activity of 94.6 µmol urea min-1 (mg protein)-1 by heat treatment and liquid chromatography methods. The enzyme, with a molecular mass of 168 kDa as determined by gel filtration, is the smallest urease known to date. Three different subunits with apparent molecular masses of 11 kDa (
or UreA; predicted molecular mass 11 kDa), 13 kDa (ß or UreB; predicted molecular mass 12 kDa) and 63 kDa (
or UreC; predicted molecular mass 62 kDa) were detected in the native enzyme, suggesting a quaternary structure of (ß)2. The Km of the purified enzyme was determined as being 0·23 mM urea. The urease activity was inhibited by HgCl2, acetohydroxamic acid and EDTA but neither by boric acid nor by L-methionine-DL-sulfoximine. Degenerate primers were designed to amplify a conserved region of the ureC gene. The amplification product was then used as a probe to clone a 5·7 kbp fragment of the P. marinus sp. strain PCC 9511 genome. The nucleotide sequence of this DNA fragment revealed two divergently orientated gene clusters, ureDABC and ureEFG, encoding the urease subunits, UreA, UreB and UreC, and the urease accessory molecules UreD, UreE, UreF and UreG. A putative NtcA-binding site was found upstream from ureEFG, indicating that this gene cluster might be under nitrogen control.
Keywords: P. marinus subsp. pastoris, Prochlorales, nitrogen metabolism, biochemical characterization, ure genes
The GenBank accession number for the sequence determined in this work is AF242489.
a Present address: Carl von Ossietzky University, ICBM, Geomicrobiology, PO Box 2503, 26111 Oldenburg, Germany.
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