The open reading frame alr1585 of Anabaena sp. strain PCC 7120 encodes a heme-dependent peroxidase (Anabaena peroxidase [AnaPX]) belonging to the novel DyP-type peroxidase family (EC 1.11.1.X). We cloned and heterologously expressed the active form of the enzyme in Escherichia coli. The purified enzyme was a 53-kDa tetrameric protein with a pI of 3.68, a low pH optima (pH 4.0), and an optimum reaction temperature of 35°C. Biochemical characterization revealed an iron protoporphyrin-containing heme peroxidase with a broad specificity for aromatic substrates such as guaiacol, 4-aminoantipyrine and pyrogallol. , respectively. In contrast, the decolorization activity of AnaPX toward azo dyes was relatively low but was significantly enhanced 2-to ϳ50-fold in the presence of the natural redox mediator syringaldehyde. The specificity and catalytic efficiency for hydrogen donors and synthetic dyes show the potential application of AnaPX as a useful alternative of horseradish peroxidase or fungal DyPs. To our knowledge, this study represents the only extensive report in which a bacterial DyP has been tested in the biotransformation of synthetic dyes.

We isolated a novel gene for cyclophilin (CyP) first identified as an intracellular target of the immunosuppressant cyclosporin A and also known to have peptidyl-prolyl cis-trans isomerase (PPIase) activity, named ATCYP5 from Arabidopsis thaliana. ATCYP5 encoded a polypeptide with 201 amino acids with a putative ER-targeting signal sequence at its N-terminal, but without the typical ER-retention signal in its C-terminal. In addition, ATCYP5 protein contained a seven amino-acid long sequence which has been found previously only in cytosolic CyPs from plants. The synthetic mutant green fluorescent protein (sGFP; S65T) was fused to the N-terminal part of ATCYP5, and expressed in tobacco BY-2 cells. The fluorescence derived from the fusion protein was detected mainly around the nucleus, indicating translocation into ER. ATCYP5 was expressed mainly in young stems especially in the apical region and weakly in leaves and roots.

Previous reports have shown that a unique bacterial dye-decolorizing peroxidase from the cyanobacterium Anabaena sp. strain PCC7120 (AnaPX) efficiently oxidizes both recalcitrant anthraquinone dyes (AQ) and typical aromatic peroxidase substrates. In this study, site-directed mutagenesis to replace five Met residues in AnaPX with high redox residues Ile, Leu, or Phe was performed for the improvement of the enzyme stability toward H(2)O(2). The heme cavity mutants M401L, M401I, M401F, and M451I had significantly increased H(2)O(2) stabilities of 2.4-, 3.7-, 8.2-, and 5.2-fold, respectively. Surprisingly, the M401F and M451I retained 16% and 5% activity at 100 mM H(2)O(2), respectively, in addition to maintaining high dye-decolorization activity toward AQ and azo dyes at 5 mM H(2)O(2) and showing a slower rate of heme degradation than the wildtype enzyme. The observed stabilization of AnaPX may be attributed to the replacement of potentially oxidizable Met residues either increasing the local stability of the heme pocket or limiting of the self-inactivation electron transfer pathways due to the above mutations. The increased stability of AnaPX variants coupled with the broad substrate specificity can be potentially useful for the further practical application of these enzymes especially in bioremediation of wastewater contaminated with recalcitrant AQ.

Gamma-glutamylcysteine synthetase (EC 6.3.2.2, gamma-GCS) catalyzes the first step of glutathione synthesis: l-Glu + l-Cys + ATP = gamma-l-glutamyl-l-cysteine (gamma-GC) + ADP + Pi. We have cloned the gene alr3351 of Anabaena sp. PCC 7120, expressed the recombinant enzyme in Escherichia coli, and characterized its product as gamma-GCS by analyzing gamma-GC production, ADP formation and Pi release. Apparent Km values for l-Glu, ATP and l-Cys were estimated to be 0.82, 0.23 and 0.14 mM, respectively. Glutathione and l-buthionine sulfoximine were inhibitors with Ki values of 6.5 and 29.3 mM, respectively. The molecular mass of Anabaena gamma-GCS was estimated to be 43.4 kDa by SDS-PAGE and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The important sequence for the activity of plant gamma-GCS was found in alpha-proteobacterial gamma-GCSs but not in cyanobacterial enzymes, suggesting that the cyanobacterial gamma-GCS gene is not the primary progenitor for the plant genes.

DyP-type peroxidases are a newly discovered family of heme peroxidases distributed from prokaryotes to eukaryotes. Recently, using a structure-based sequence alignment, we proposed the new classes, P, I and V, as substitutes for classes A, B, C, and D [Arch Biochem Biophys 2015;574:49-55]. Although many class V enzymes from eukaryotes have been characterized, only two from prokaryotes have been reported. Here, we show the crystal structure of one of these two enzymes, Anabaena sp. DyP-type peroxidase (AnaPX). AnaPX is tetramer formed from Cys224-Cys224 disulfide-linked dimers. The tetramer of wild-type AnaPX was stable at all salt concentrations tested. In contrast, the C224A mutant showed salt concentration-dependent oligomeric states: in 600 mM NaCl, it maintained a tetrameric structure, whereas in the absence of salt, it dissociated into monomers, leading to a reduction in thermostability. Although the tetramer exhibits non-crystallographic, 2-fold symmetry in the asymmetric unit, two subunits forming the Cys224-Cys224 disulfide-linked dimer are related by 165° rotation. This asymmetry creates an opening to cavities facing the inside of the tetramer, providing a pathway for hydrogen peroxide access. Finally, a phylogenetic analysis using structure-based sequence alignments showed that class V enzymes from prokaryotes, including AnaPX, are phylogenetically closely related to class V enzymes from eukaryotes.