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J. Biol. Chem., Vol. 277, Issue 28, 25096-25105, July 12, 2002

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Isolation of Cyanophycin-degrading Bacteria, Cloning and Characterization of an Extracellular Cyanophycinase Gene (cphE) from Pseudomonas anguilliseptica Strain BI
THE cphE GENE FROM P. ANGUILLISEPTICA BI ENCODES A CYANOPHYCIN-HYDROLYZING ENZYME^*

Martin Obst, Fred Bernd Oppermann-Sanio, Heinrich Luftmann^§, and Alexander Steinbüchel^¶

From the  Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 3, D-48149 Münster, Germany and the ^§ Institut für Organische Chemie, Westfälische Wilhelms-Universität Münster, Corrensstrasse 40, D-48149 Münster, Germany

Eleven bacteria capable of utilizing cyanophycin (cyanophycin granule polypeptide (CGP)) as a carbon source for growth were isolated. One isolate was taxonomically affiliated as Pseudomonas anguilliseptica strain BI, and the extracellular cyanophycinase (CphE) was studied because utilization of cyanophycin as a carbon source and extracellular cyanophycinases were hitherto not described. CphE was detected in supernatants of CGP cultures and purified from a corresponding culture of strain BI employing chromatography on the anion exchange matrix Q-Sepharose and on an arginine-agarose affinity matrix. The mature form of the inducible enzyme consisted of one type of subunit with M_r = 43,000 and exhibited high specificity for CGP, whereas proteins and synthetic polyaspartic acid were not hydrolyzed or were only marginally hydrolyzed. Degradation products of the enzyme reaction were identified as aspartic acid-arginine dipeptides (-Asp-Arg) by high performance liquid chromatography and electrospray ionization mass spectrometry. The corresponding gene (cphE, 1254 base pairs) was identified in subclones of a cosmid gene library of strain BI by heterologous active expression in Escherichia coli, and its nucleotide sequence was determined. The enzyme exhibited only 27-28% amino acid sequence identity to intracellular cyanophycinases occurring in cyanobacteria. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the motif GXSXG plus a histidine and most probably a glutamate residue. In addition, the strong inhibition of the enzyme by Pefabloc^® and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases. Quantitative analysis on the release of -Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism.

The amino acid sequence(s) of this protein can be accessed through NCBI Protein Database under NCBI accession numbers AF439803 and AY065671.

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