Abstract
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 (beta-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((R)) and phenylmethylsulfonyl fluoride indicated that the catalytic mechanism of CphE is related to that of serine type proteases. Quantitative analysis on the release of beta-Asp-Arg dipeptides from C-terminal labeled CGP gave evidence for an exo-degradation mechanism.
Highlights
Cyanophycin is a natural occurring poly(amino acid), which is synthesized in most cyanobacteria as nitrogen, carbon and energy storage compound in the early stationary growth phase [1,2]
Most of the β-carboxylic groups are covalently bound to the α-amino groups of L-arginine residues [4,5]; in recombinant Escherichia coli expressing cyanobacterial CGP synthesizing enzymes a significant fraction of arginine is replaced by lysine [6]
Whereas much information has been obtained concerning the non-ribosomal biosynthesis of CGP, which is catalysed by the cyanophycin synthetase (CphA, [7] and cited references therein), only few reports are available on the intracellular degradation of CGP
Summary
Cyanophycin (cyanophycin granule polypeptide, CGP) is a natural occurring poly(amino acid), which is synthesized in most cyanobacteria as nitrogen, carbon and energy storage compound in the early stationary growth phase [1,2]. The water insoluble CGP is accumulated intracellularly in the form of membraneless granules [3] and is degraded by the cells when growth is resumed. The backbone of this unique biopolymer consists of α-amino-α-carboxy-linked L-aspartic acid monomers. Most of the β-carboxylic groups are covalently bound to the α-amino groups of L-arginine residues [4,5]; in recombinant Escherichia coli expressing cyanobacterial CGP synthesizing enzymes (see below) a significant fraction of arginine is replaced by lysine [6]. In contrast to intracellular degradation, nothing is known about the extracellular decomposition of this biopolymer by bacteria or other microorganisms
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