The lack of rhodanese RhdA affects the sensitivity of Azotobacter vinelandii to oxidative events

The rhdA gene of Azotobacter vinelandii codes for RhdA, a rhodanese-domain protein with an active-site loop structure which has not currently been found in proteins of the rhodanese-homology superfamily. Considering the lack of information on the functional role of the ubiquitous rhodaneses, in the present study we examined the in vivo functions of RhdA by using an A. vinelandii mutant strain (MV474), in which the rhdA gene was disrupted by deletion. Preliminary phenotypic characterization of the rhdA mutant suggested that RhdA could exert protection over Fe-S enzymes, which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe-S clusters, in the present study we analysed the defects of the rhdA-null strain by exploiting growth conditions which resulted in enhancing the catalytic deficiency of enzymes with vulnerable Fe-S clusters. We found that a lack of RhdA impaired A. vinelandii growth in the presence of gluconate, a carbon source that activates the Entner-Doudoroff pathway in which the first enzyme, 6-phosphogluconate dehydratase, employs a 4Fe-4S cluster as an active-site catalyst. By combining proteomics, enzymatic profiles and model systems to generate oxidative stress, evidence is provided that to rescue the effects of a lack of RhdA, A. vinelandii needed to activate defensive activities against oxidative damage. The possible functionality of RhdA as a redox switch which helps A. vinelandii in maintaining the cellular redox balance was investigated by using an in vitro model system that demonstrated reversible chemical modifications in the highly reactive RhdA Cys(230) thiol.     

Mutagenic analysis of Thr-232 in rhodanese from Azotobacter vinelandii highlighted the differences of this prokaryotic enzyme from the known sulfurtransferases

Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys-249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate-cyanide sulfurtransferase activity, and no detectable activity in the presence of 3-mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase.     

The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form

After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified l-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA.     

Evidence that elongation of the catalytic loop of the Azotobacter vinelandii rhodanese changed selectivity from sulfur- to phosphate-containing substrates

Recent investigations have shown that the rhodanese domains, ubiquitous structural modules which might represent an example of conserved structures with possible functional diversity, are structurally related to the catalytic subunit of Cdc25 phosphatase enzymes. The major difference characterizing the active-site of the Azotobacter vinelandii rhodanese RhdA, with respect to the closely related Cdc25s (A, B, C), is that in Cdc25 phosphatases the active site loop [His-Cys-(X)5-Arg] is one residue longer than in RhdA [His-Cys-(X)4-Arg]. According to the hypothesis that the length of the RhdA active-site loop should play a key role in substrate recognition and catalytic activity, RhdA scaffold was the starting point for producing mutants with single-residue insertion to generate the catalytic loop HCQTHAHR (in RhdA-Ala) and HCQTHSHR (in RhdA-Ser). Analyses of the catalytic performances of the engineered RhdAs revealed that elongation of the catalytic loop definitely compromised the ability to catalyze sulfur transfer reactions, while it generated 'phosphatase' enzymes able to interact productively with the artificial substrate 3-O-methylfluorescein phosphate. Although this study is restricted to an example of rhodanese modules (RhdA), it provided experimental evidence of the hypothesis that a specific mutational event (a single-residue insertion or deletion in the active-site loop) could change the selectivity from sulfur- to phosphate-containing substrates (or vice versa).     

Involvement of the Azotobacter vinelandii Rhodanese-Like Protein RhdA in the Glutathione Regeneration Pathway

The phenotypic features of the Azotobacter vinelandii RhdA mutant MV474 (in which the rhdA gene was deleted) indicated that defects in antioxidant systems in this organism were related to the expression of the tandem-domain rhodanese RhdA. In this work, further insights on the effects of the oxidative imbalance generated by the absence of RhdA (e.g. increased levels of lipid hydroperoxides) are provided. Starting from the evidence that glutathione was depleted in MV474, and using both in silico and in vitro approaches, here we studied the interaction of wild-type RhdA and Cys²³⁰Ala site-directed RhdA mutant with glutathione species. We found that RhdA was able to bind in vitro reduced glutathione (GSH) and that RhdA-Cys²³⁰ residue was mandatory for the complex formation. RhdA catalyzed glutathione-disulfide formation in the presence of a system generating the glutathione thiyl radical (GS^•, an oxidized form of GSH), thereby facilitating GSH regeneration. This reaction was negligible when the Cys²³⁰Ala RhdA mutant was used. The efficiency of RhdA as catalyst in GS^•-scavenging activity is discussed on the basis of the measured parameters of both interaction with glutathione species and kinetic studies.     

A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii Rhodanese

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, site-directed mutagenesis, and X-ray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common three-dimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.     

Characterization of a rhodanese from the cyanogenic bacterium Pseudomonas aeruginosa

Pseudomonas aeruginosa, the rRNA group I type species of genus Pseudomonas, is a Gram-negative, aerobic bacterium responsible for serious infection in humans. P. aeruginosa pathogenicity has been associated with the production of several virulence factors, including cyanide. Here, the biochemical characterization of recombinant P. aeruginosa rhodanese (Pa RhdA), catalyzing the sulfur transfer from thiosulfate to a thiophilic acceptor, e.g., cyanide, is reported. Sequence homology analysis of Pa RhdA predicts the sulfur-transfer reaction to occur through persulfuration of the conserved catalytic Cys230 residue. Accordingly, the titration of active Pa RhdA with cyanide indicates the presence of one extra sulfur bound to the Cys230 Sgamma atom per active enzyme molecule. Values of K(m) for thiosulfate binding to Pa RhdA are 1.0 and 7.4mM at pH 7.3 and 8.6, respectively, and 25 degrees C. However, the value of K(m) for cyanide binding to Pa RhdA (=14 mM, at 25 degrees C) and the value of V(max) (=750 micromol min(-1)mg(-1), at 25 degrees C) for the Pa RhdA-catalyzed sulfur-transfer reaction are essentially pH- and substrate-independent. Therefore, the thiosulfate-dependent Pa RhdA persulfuration is favored at pH 7.3 (i.e., the cytosolic pH of the bacterial cell) rather than pH 8.6 (i.e., the standard pH for rhodanese activity assay). Within this pH range, conformational change(s) occur at the Pa RhdA active site during the catalytic cycle. As a whole, rhodanese may participate in multiple detoxification mechanisms protecting P. aeruginosa from endogenous and environmental cyanide.     

The crystal structure of a sulfurtransferase from Azotobacter vinelandii highlights the evolutionary relationship between the rhodanese and phosphatase enzyme families

Rhodanese is an ubiquitous enzyme that in vitro catalyses the transfer of a sulfur atom from suitable donors to nucleophilic acceptors by way of a double displacement mechanism. During the catalytic process the enzyme cycles between a sulfur-free and a persulfide-containing form, via formation of a persulfide linkage to a catalytic Cys residue. In the nitrogen-fixing bacteria Azotobacter vinelandii the rhdA gene has been identified and the encoded protein functionally characterized as a rhodanese. The crystal structure of the A. vinelandii rhodanese has been determined and refined at 1.8 A resolution in the sulfur-free and persulfide-containing forms. Conservation of the overall three-dimensional fold of bovine rhodanese is observed, with substantial modifications of the protein structure in the proximity of the catalytic residue Cys230. Remarkably, the native enzyme is found as the Cys230-persulfide form; in the sulfur-free state the catalytic Cys residue adopts two alternate conformations, reflected by perturbation of the neighboring active-site residues, which is associated with a partly reversible loss of thiosulfate:cyanide sulfurtransferase activity. The catalytic mechanism of A. vinelandii rhodanese relies primarily on the main-chain conformation of the 230 to 235 active-site loop and on a surrounding strong positive electrostatic field. Substrate recognition is based on residues which are entirely different in the prokaryotic and eukaryotic enzymes. The active-site loop of A. vinelandii rhodanese displays striking structural similarity to the active-site loop of the similarly folded catalytic domain of dual specific phosphatase Cdc25, suggesting a common evolutionary origin of the two enzyme families.     

Molecular recognition between Azotobacter vinelandii rhodanese and a sulfur acceptor protein

The occurrence of rhodanese-like proteins in the major evolutionary phyla, together with the observed abundance of these proteins also within the same genome, suggests that their function cannot be limited to cyanide scavenging. The aim of the present study was to investigate whether Azotobacter vinelandii RhdA, an enzyme possessing unique biochemical and structural features with respect to other members of rhodanese homology superfamily, could recognize a suitable protein as a potential acceptor of the sulfane sulfur held on its catalytic Cys residue. Both the potential sulfur-delivery RhdA-S and the sulfur-deprived RhdA were found to interact with either holo- or apo-adrenodoxin, the 'substrate' protein used in this work. Interaction of RhdA-S with apo-adrenodoxin led to mobilization of RhdA-S sulfane sulfur. Under appropriate conditions, the sulfur released from RhdA-S was productively used for 2Fe-2S cluster reconstitution to yield holo-adrenodoxin from apo-adrenodoxin in the absence of any other sulfur source. A comparison of the reactivity of RhdA-S with protein and non-protein thiols allowed also some insights into the accessibility of the sulfane sulfur carried by RhdA.     

Effects of chronic sub-lethal oxidative stress on biofilm formation by Azotobacter vinelandii

This work showed that perturbations of the physiological steady-state level of reactive oxygen species (ROS) affected biofilm genesis and the characteristics of the model bacterium Azotobacter vinelandii. To get a continuous endogenous source of ROS, a strain exposed to chronic sub-lethal oxidative stress was deprived of the gene coding for the antioxidant rhodanese-like protein RhdA (MV474). In this study MV474 biofilm showed (i) a seven-fold higher growth rate, (ii) induction of catalase and alkyl-hydroxyl-peroxidase enzymes, (iii) higher average thicknesses due to increased production of a polysaccharide-rich extracellular matrix and (iv) less susceptibility to hydrogen peroxide than the wild-type strain (UW136). MV474 showed increased swimming and swarming activity and the swarming colonies experienced a higher level of oxidative stress compared to UW136. A continuous exogenous source of ROS increased biofilm formation in UW136. Overall, chronic sub-lethal oxidative events promoted sessile behavior in A. vinelandii.     

Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α-l-arabinofuranosidase 54

A role for N-linked oligosaccharides on the biochemical properties of recombinant α-l-arabinofuranosidase 54 (AkAbf54) defined in glycoside hydrolase family 54 from Aspergillus kawachii expressed in Pichia pastoris was analyzed by site-directed mutagenesis. Two N-linked glycosylation motifs (Asn⁸³–Thr–Thr and Asn²⁰²–Ser–Thr) were found in the AkAbf54 sequence. AkAbf54 comprises two domains, a catalytic domain and an arabinose-binding domain classified as carbohydrate-binding module 42. Two N-linked glycosylation sites are located in the catalytic domain. Asn⁸³, Asn²⁰², and the two residues together were replaced with glutamine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the wild-type and mutant enzymes expressed in P. pastoris were examined. The N83Q mutant enzyme had the same catalytic activity and thermostability as the wild-type enzyme. On the other hand, the N202Q and N83Q/N202Q mutant enzymes exhibited a considerable decrease in thermostability compared to the glycosylated wild-type enzyme. The N202Q and N83Q/N202Q mutant enzymes also had slightly less specific activity towards arabinan and debranched arabinan. However, no significant effect on the affinity of the mutant enzymes for the ligands arabinan, debranched arabinan, and wheat and rye arabinoxylans was detected by affinity gel electrophoresis. These observations suggest that the glycosylation at Asn²⁰² may contribute to thermostability and catalysis.     

Retinoic acid-metabolizing enzyme Cyp26a1 is essential for determining territories of hindbrain and spinal cord in zebrafish

Retinoic acid (RA) plays a critical role in neural patterning and organogenesis in the vertebrate embryo. Here we characterize a mutant of the zebrafish named giraffe (gir) in which the gene for the RA-degrading enzyme Cyp26a1 is mutated. The gir mutant displayed patterning defects in multiple organs including the common cardinal vein, pectoral fin, tail, hindbrain, and spinal cord. Analyses of molecular markers suggested that the lateral plate mesoderm is posteriorized in the gir mutant, which is likely to cause the defects of the common cardinal vein and pectoral fin. The cyp26a1 expression in the rostral spinal cord was strongly upregulated in the gir mutant, suggesting a strong feedback control of its expression by RA signaling. We also found that the rostral spinal cord territory was expanded at the expense of the hindbrain territory in the gir mutant. Such a phenotype is the opposite of that of the mutant for Raldh2, an enzyme that synthesizes RA. We propose a model in which Cyp26a1 attenuates RA signaling in the prospective rostral spinal cord to limit the expression of hox genes and to determine the hindbrain-spinal cord boundary.     

Respiratory-competent conditional developmental mutant of Mucor racemosus

A conditional developmental mutant of Mucor racemosus which is capable of oxidative energy metabolism is described. Unlike the wild-type strain the mutant was highly fermentative and exhibited the yeast morphology when grown aerobically in glucose-containing media. The high fermentative activity and yeast morphology under these conditions correlated well with maximal expression of glycolytic enzymes and with expression of some polypeptides characteristic of anaerobic growth. Aerobic growth of the mutant on amino acids as the sole carbon source resulted in growth in the mycelial morphology. The mutant was fully capable of oxidative metabolism as judged by its ability to grow on amino acids, respiratory capacity, and complement of tricarboxylic acid cycle enzymes. The results support the hypothesis that oxygen controls both the expression of glycolytic enzymes and the expression of proteins involved in morphogenesis. Moreover, they suggest that there are common regulatory elements in the control of these two classes of gene products. Abnormally high levels of aconitase and isocitrate dehydrogenase in the mutant are consistent with the proposal that pool sizes of citrate may act as a regulator of genes responsive to environmental oxygen concentration.