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Background: The exceptional capacities of aquaporins in terms of water permeation and selectivity have made them an interesting system for membrane applications. Despite the multiple attempts for immobilizing the aquaporins over a porous substrate, there is a lack of studies related to the purification and reconstitution steps, principally associated with the use of detergents in solubilization and destabilization steps. This study analyzed the effect of detergents in Aquaporin Z solubilization, considering the purity and structural homogeneity of the protein., Methods: The extraction process was optimized by the addition of detergent at the sonication step, which enabled the omission of the ultracentrifugation and resuspension steps. Two detergents, Triton X-100, and octyl-glucoside were also evaluated. Destabilization mediated by detergents was used as reconstitution method. Saturation and solubilization points were defined by detergent concentration and both, liposomes and proteoliposomes, were analyzed by size distribution and permeability assays. Detergent removal with Bio-beads was also analyzed., Results: Octyl glucoside ensures structural stability and homogeneity of Aquaporin Z. However, high concentrations of detergents induce the presence of defects in proteoliposomes. While saturated liposomes create homogeneous and functional structures, solubilized liposomes get affected by a reassembly process, creating vesicle defects with anomalous permeability profiles., Conclusions: Detergent concentration affects the structural conformation of proteoliposomes in the reconstitution process., General Significance: Since the destabilization process is dependent on vesicle, detergent, and buffer composition, optimization of this process should be mandatory for further studies. All these considerations will allow achieving the potential of Aquaporins and any other integral membrane protein in their applications for industrial purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea. This enzyme has several analogies with agmatinase, which catalyzes the hydrolysis of agmatine into putrescine and urea. However, this contrasts with the highlighted specificity that each one presents for their respective substrate. A comparison of available crystal structures for arginases reveals an important difference in the extension of two loops located in the entrance of the active site. The first, denominated loop A (I129-L140) contains the residues that interact with the alpha carboxyl group or arginine of arginase, and the loop B (D181-P184) contains the residues that interact with the alpha amino group of arginine. In this work, to determine the importance of these loops in the specificity of arginase, single, double, and triple arginase mutants in these loops were constructed, as well as chimeras between type I human arginase and E. coli agmatinase. In previous studies, the substitution of N130D in arginase (in loop A ) generated a species capable of hydrolyzing arginine and agmatine. Now, the specificity of arginase is completely altered, generating a chimeric species that is only active with agmatine as a substrate, by substituting I129T, N130Y, and T131A together with the elimination of residues P132, L133, and T134. In addition, Quantum Mechanic/Molecular Mechanic (QM/MM) calculations were carried out to study the accommodation of the substrates in in the active site of this chimera. With these results it is concluded that this loop is decisive to discriminate the type of substrate susceptible to be hydrolyzed by arginase. Evidence was also obtained to define the loop B as a structural determinant for substrate affinity. Concretely, the double mutation D181T and V182E generate an enzyme with an essentially unaltered k cat value, but with a significantly increased K m value for arginine and a significant decrease in affinity for its product ornithine.

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer's, Parkinson's, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn 2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

Cryptosporidiosis, caused by protozoan parasites of the genus Cryptosporidium, is estimated to rank as a leading cause in the global burden of neglected zoonotic parasitic diseases. This diarrheal disease is the second leading cause of death in children under 5 years of age. Based on the C. parvum transcriptome data, glutathione transferase (GST) has been suggested as a drug target against this pathogen. GSTs are diverse multifunctional proteins involved in cellular defense and detoxification in organisms and help pathogens to alleviate chemical and environmental stress. In this study, we performed genome-wide data mining, identification, classification and in silico structural analysis of GSTs in fifteen Cryptosporidium species. The study revealed the presence three GSTs in each of the Cryptosporidium species analyzed in the study. Based on the percentage identity and comprehensive comparative phylogenetic analysis, we assigned Cryptosporidium species GSTs to three new GST classes, named Vega (ϑ), Gamma (γ) and Psi (ψ). The study also revealed an atypical thioredoxin-like fold in the C. parvum GST1 of the Vega class, whereas C. parvum GST2 of the Gamma class and C. melagridis GST3 of the Psi class has a typical thioredoxin-like fold in the N-terminal region. This study reports the first comparative analysis of GSTs in Cryptosporidium species.

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins that are ubiquitously present in organisms, including non-living entities such as viruses. With the exception of self-sufficient P450s, all other P450 enzymes need electrons to perform their enzymatic activity and these electrons are supplied by P450 redox proteins. Different types of P450 redox proteins can be found in organisms and are classified into different classes. Bacterial P450s (class I) receive electrons from ferredoxins which are iron-sulfur cluster proteins. The presence of more than one copy and different types of ferredoxins within a bacterial species poses fundamental questions about the selectivity of P450s and ferredoxins in relation to each other. Apart from transferring electrons, ferredoxins have also been found to modulate P450 functions. Achieving an understanding of the interaction between ferredoxins and P450s is required to harness their biotechnological potential for designing a universal electron transfer protein. A brief overview of factors playing a role in ferredoxin and P450 interactions is presented in this review article.

Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. ALP, despite differing in amino acid sequence from all members of the ureohydrolase family, is strictly dependent on Mn 2+ for catalytic activity. However, the Mn 2+ ligands have not yet been identified due to the lack of structural information coupled with the low sequence identity that ALPs display with known ureohydrolases. In this work, we generated a structural model of the Mn 2+ binding site of the ALP and we propose new putative Mn 2+ ligands. Then, we cloned and expressed a sequence of 210 amino acids, here called the "central-ALP", which include the putative ligands of Mn 2+ . The results suggest that the central-ALP is catalytically active, as agmatinase, with an unaltered K m for agmatine and a decreased k cat . Similar to wild-type ALP, central-ALP is activated by Mn 2+ with a similar affinity. Besides, a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of these putative Mn 2+ ligands result on the loss of ALP agmatinase activity. Our results indicate that the central-ALP contains the active site for agmatine hydrolysis, as well as that the residues identified are relevant for the ALP catalysis.

Phycobiliproteins (PBPs) are colored fluorescent proteins present in cyanobacteria, red alga, and cryptophyta. These proteins have many potential uses in biotechnology going from food colorants to medical applications. Allophycocyanin, the simplest PBP, is a heterodimer of αβ subunits that oligomerizes as a trimer (αβ) 3 . Each subunit contains a phycocyanobilin, bound to a cysteine residue, which is responsible for its spectroscopic properties. In this article, we are reporting the expression of recombinant allophycocyanin (rAPC) from the eukaryotic red algae Agarophyton chilensis in Escherichia coli, using prokaryotic accessory enzymes to obtain a fully functional rAPC. Three duet vectors were used to include coding sequences of α and β subunits from A. chilensis and accessorial enzymes (heterodimeric lyase cpc S/U, heme oxygenase 1, phycocyanobilin oxidoreductase) from cyanobacteria Arthrospira maxima. rAPC was purified using several chromatographic steps. The characterization of the pure rAPC indicates very similar spectroscopic properties, λ max Abs , λ max Em , fluorescence lifetime, and chromophorylation degree, with native allophycocyanin (nAPC) from A. chilensis. This method, to produce high-quality recombinant allophycocyanin, can be used to express and characterize other macroalga phycobiliproteins, to be used for biotechnological or biomedical purposes., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

[This corrects the article DOI: 10.1371/journal.pone.0195656.].

Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to Photosystems due to chromophores covalently bound to specific cysteine residues. Besides phycobiliproteins (PE), the PBS contains linker proteins responsible for assembly and stabilization of the whole complex and the tuning of energy transfer steps between chromophores. The linker (γ33) from Gracilaria chilensis, is a chromophorylated rod linker associated to (αβ)6 hexamers of R-phycoerythrin (R-PE). Its role in the energy transfer process is not clear yet. Structural studies as well as the composition and location of the chromophores are essential to understand their involvement in the energy transfer process in PBS. To achieve this, the coding gene of γ33 was cloned and sequenced. The sequence was analyzed by informatics tools, to obtain preliminary information which leaded the next experiments. The protein was purified from R-phycoerythrin, and the sequence confirmed by mass spectrometry. The coding sequence analysis revealed a protein of 318 aminoacid residues containing a chloroplastidial transit peptide (cTP) of 39 aminoacids at the N-terminus. The conservation of cysteines revealed possible chromophorylation sites. Using α and β R-PE subunits as spectroscopic probes in denaturation assays, we deduced a double bonded phycourobilin (PUB) on γ33 subunit that were confirmed between Cys62 and Cys73 (DL-PUB62/73) by mass spectrometry. The cysteines involved in the double link are located in a helical region, in a conformation that reminds the position of the DL-PUB50/61 in the β subunit of R-PE. The position of single linked PUB at Cys95 and a single linked PEB at Cys172 were also confirmed. Spectroscopic studies show the presence of both types of chromophores and that there are not energy transfer by FRET among them.

Backgroud: Ferredoxin NADP(H) oxidoreductases (EC 1.18.1.2) (FNR) are flavoenzymes present in photosynthetic organisms; they are relevant for the production of reduced donors to redox reactions, i.e. in photosynthesis, the reduction of NADP + to NADPH using the electrons provided by Ferredoxin (Fd), a small FeS soluble protein acceptor of electrons from PSI in chloroplasts. In rhodophyta no information about this system has been reported, this work is a contribution to the molecular and functional characterization of FNR from Gracilaria chilensis, also providing a structural analysis of the complex FNR/Fd., Methods: The biochemical and kinetic characterization of FNR was performed from the enzyme purified from phycobilisomes enriched fractions. The sequence of the gene that codifies for the enzyme, was obtained using primers designed by comparison with sequences of Synechocystis and EST from Gracilaria. 5'RACE was used to confirm the absence of a CpcD domain in FNRPBS of Gracilaria chilensis. A three dimensional model for FNR and Fd, was built by comparative modeling and a model for the complex FNR: Fd by docking., Results: The kinetic analysis shows K M NADPH of 12.5 M and a k cat of 86 s -1 , data consistent with the parameters determined for the enzyme purified from a soluble extract. The sequence for FNR was obtained and translated to a protein of 33646 Da. A FAD and a NADP+ binding domain were clearly identified by sequence analysis as well as a chloroplast signal sequence. Phycobilisome binding domain, present in some cyanobacteria was absent. Transcriptome analysis of Gch revealed the presence of two Fd; FdL and FdS , sharing the motif CX5CX2CX29X. The analysis indicated that the most probable partner for FNR is FdS., Conclusion: The interaction model produced, was consistent with functional properties reported for FNR in plants leaves, and opens the possibilities for research in other rhodophyta of commercial interest.

Phycobilisomes (PBS) are accessory light harvesting protein complexes that directionally transfer energy towards photosystems. Phycobilisomes are organized in a central core and rods radiating from it. Components of phycobilisomes in Gracilaria chilensis (Gch) are Phycobiliproteins (PBPs), Phycoerythrin (PE), and Phycocyanin (PC) in the rods, while Allophycocyanin (APC) is found in the core, and linker proteins (L). The function of such complexes depends on the structure of each component and their interaction. The core of PBS from cyanobacteria is mainly composed by cylinders of trimers of α and β subunits forming heterodimers of Allophycocyanin, and other components of the core including subunits αII and β18. As for the linkers, Linker core (LC) and Linker core membrane (LCM) are essential for the final emission towards photoreaction centers. Since we have previously focused our studies on the rods of the PBS, in the present article we investigated the components of the core in the phycobilisome from the eukaryotic algae, Gracilaria chilensis and their organization into trimers. Transmission electron microscopy provided the information for a three cylinders core, while the three dimensional structure of Allophycocyanin purified from Gch was determined by X-ray diffraction method and the biological unit was determined as a trimer by size exclusion chromatography. The protein sequences of all the components of the core were obtained by sequencing the corresponding genes and their expression confirmed by transcriptomic analysis. These subunits have seldom been reported in red algae, but not in Gracilaria chilensis. The subunits not present in the crystallographic structure were modeled to build the different composition of trimers. This article proposes structural models for the different types of trimers present in the core of phycobilisomes of Gch as a first step towards the final model for energy transfer in this system.

Energy transfer (ET) in phycobilisomes, a macrocomplex of phycobiliproteins and linker proteins, is a process that is difficult to understand completely. A model for a rod composed of two hexamers of Phycocyanin and two hexamers of Phycoerythrin was built using an in silico approach and the three-dimensional structures of both phycobiliproteins from Gracilaria chilensis. The model was characterized and showed 125 Å wide and 230 Å high, which agree with the dimensions of a piling of four hexamers as observed in the images of subcomplexes of phycobilisomes obtained by transmission electron microscopy. ET rates between every pair of chromophores in the model were calculated using the Förster approach, and the fastest rates were selected to draw preferential ET pathways along the rod. Every path indicates that the ET is funneled toward the chromophores located at Cysteines 82 in Phycoerythrin and 84 in Phycocyanin. The chromophores that face the exterior of the rod are phycoerythrobilins, and they also show a preferential ET toward the chromophores located at the center of the rod. The values calculated, in general, agree with the experimental data reported previously, which validates the use of this experimental approach., (Copyright © 2012 The Protein Society.)