Insights into the mechanism and regulation of the CbbQO-type Rubisco activase, a MoxR AAA+ ATPase.

The vast majority of biological carbon dioxide fixation relies on the function of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). In most cases the enzyme exhibits a tendency to become inhibited by its substrate RuBP and other sugar phosphates. The inhibition is counteracted by diverse molecular chaperones known as Rubisco activases (Rcas). In some chemoautotrophic bacteria, the CbbQO-type Rca Q2O2 repairs inhibited active sites of hexameric form II Rubisco. The 2.2-Å crystal structure of the MoxR AAA+ protein CbbQ2 from Acidithiobacillus ferrooxidans reveals the helix 2 insert (H2I) that is critical for Rca function and forms the axial pore of the CbbQ hexamer. Negative-stain electron microscopy shows that the essential CbbO adaptor protein binds to the conserved, concave side of the CbbQ2 hexamer. Site-directed mutagenesis supports a model in which adenosine 5'-triphosphate (ATP)-powered movements of the H2I are transmitted to CbbO via the concave residue L85. The basal ATPase activity of Q2O2 Rca is repressed but strongly stimulated by inhibited Rubisco. The characterization of multiple variants where this repression is released indicates that binding of inhibited Rubisco to the C-terminal CbbO VWA domain initiates a signal toward the CbbQ active site that is propagated via elements that include the CbbQ α4-ß4 loop, pore loop 1, and the presensor 1-ß hairpin (PS1-ßH). Detailed mechanistic insights into the enzyme repair chaperones of the highly diverse CO2 fixation machinery of Proteobacteria will facilitate their successful implementation in synthetic biology ventures.

Bacterial Respiratory Chain Diversity Reveals a Cytochrome c Oxidase Reducing O2 at Low Overpotentials.

Cytochrome c oxidases (CcOs) are the terminal enzymes in energy-converting chains of microorganisms, where they reduce oxygen into water. Their affinity for O2 makes them attractive biocatalysts for technological devices in which O2 concentration is limited, but the high overpotentials they display on electrodes severely limit their applicative use. Here, the CcO of the acidophilic bacterium Acidithiobacillus ferrooxidans is studied on various carbon materials by direct protein electrochemistry and mediated one with redox mediators either diffusing or co-immobilized at the electrode surface. The entrapment of the CcO in a network of hydrophobic carbon nanofibers permits a direct electrochemical communication between the enzyme and the electrode. We demonstrate that the CcO displays a µM affinity for O2 and reduces O2 at exceptionally high electrode potentials in the range of +700 to +540 mV vs NHE over a pH range of 4-6. The kinetics of interactions between the enzyme and its physiological partners are fully quantified. Based on these results, an electron transfer pathway allowing O2 reduction in the acidic metabolic chain is proposed.

Iron and sulfur oxidation pathways of Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a gram-negative, autotrophic and rod-shaped bacterium. It can gain energy through the oxidation of Fe(II) and reduced inorganic sulfur compounds for bacterial growth when oxygen is sufficient. It can be used for bio-leaching and bio-oxidation and contributes to the geobiochemical circulation of metal elements and nutrients in acid mine drainage environments. The iron and sulfur oxidation pathways of A. ferrooxidans play key roles in bacterial growth and survival under extreme circumstances. Here, the electrons transported through the thermodynamically favourable pathway for the reduction to H2O (downhill pathway) and against the redox potential gradient reduce to NAD(P)(H) (uphill pathway) during the oxidation of Fe(II) were reviewed, mainly including the electron transport carrier, relevant operon and regulation of its expression. Similar to the electron transfer pathway, the sulfur oxidation pathway of A. ferrooxidans, related genes and operons, sulfur oxidation mechanism and sulfur oxidase system are systematically discussed.

Stabilization of multimeric sucrose synthase from Acidithiobacillus caldus via immobilization and post-immobilization techniques for synthesis of UDP-glucose.

Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5'-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 °C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.

Characterization of tetrathionate hydrolase from the marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH.

Tetrathionate hydrolase (4THase), a key enzyme of the S4-intermediate (S4I) pathway, was partially purified from marine acidophilic bacterium, Acidithiobacillus thiooxidans strain SH, and the gene encoding this enzyme (SH-tth) was identified. SH-Tth is a homodimer with a molecular mass of 97 ± 3 kDa, and contains a subunit 52 kDa in size. Enzyme activity was stimulated in the presence of 1 M NaCl, and showed the maximum at pH 3.0. Although 4THases from A. thiooxidans and the closely related Acidithiobacillus caldus strain have been reported to be periplasmic enzymes, SH-Tth seems to be localized on the outer membrane of the cell, and acts as a peripheral protein. Furthermore, both 4THase activity and SH-Tth proteins were detected in sulfur-grown cells of strain SH. These results suggested that SH-Tth is involved in elemental sulfur-oxidation, which is distinct from sulfur-oxidation in other sulfur-oxidizing strains such as A. thiooxidans and A. caldus.

Interplay Between Expression of Sulfur Assimilation Pathway Genes and Zn(2+) and Pb(2+) Stress in Acidithiobacillus ferrooxidans.

We have previously demonstrated that in Acidithiobacillus ferrooxidans, resistance to the highly toxic divalent cation Cd(2+) is mediated in part by the sulfur assimilation pathway (SAP) and enhanced intracellular concentrations of cysteine and glutathione(GSH) (Zheng et al., Extremophiles 19:429-436, 2015). In this paper, we investigate the interplay between Zn(2+) and Pb(2+) resistances, SAP gene expression, and thiol-containing metabolite levels. Cells grown in the presence of 300 mM Zn(2+) had enhanced activities of the following enzymes: adenosylphosphosulphate reductase (APR, 40-fold), serine acetyltransferase (SAT, 180-fold), and O-acetylserine (thiol) lyase (OAS-TL, 230-fold). We investigated the concentrations of mRNA transcripts of the genes encoding these enzymes in cells grown in the presence of 600 mM Zn(2+): transcripts for 4 SAP genes-ATPS(ATP sulphurylase), APR, SiR(sulfite reductase), SAT, and OAS-TL-each showed a more than three-fold increase in concentration. At the metabolite level, concentrations of intracellular cysteine and glutathione (GSH) were nearly doubled. When cells were grown in the presence of 10 mM Pb(2+), SAP gene transcript concentrations, cysteine, and GSH concentrations were all decreased, as were SAP enzyme activities. These results suggested that Zn(2+) induced SAP pathway gene transcription, while Pb(2+) inhibited SAP gene expression and enzyme activities compared to the pathway in most organisms. Because of the detoxification function of thiol pool, the results also suggested that the high resistance of A. ferrooxidans to Zn(2+) may also be due to regulation of GSH and the cysteine synthesis pathway.

[Phylogenetic and diversity analysis of Acidithiobacillus spp. based on 16S rRNA and RubisCO genes homologues].

Objective: The purpose of the study was to reveal geographic region-related Acidithiobacillus spp. distribution and allopatric speciation. Phylogenetic and diversity analysis was done to expand our knowledge on microbial phylogeography, diversity-maintaining mechanisms and molecular biogeography. Methods: We amplified 16S rRNA gene and RubisCO genes to construct corresponding phylogenetic trees based on the sequence homology and analyzed genetic diversity of Acidithiobacillus spp.. Results: Thirty-five strains were isolated from three different regions in China (Yunnan, Hubei, Xinjiang). The whole isolates were classified into five groups. Four strains were identified as A. ferrivorans, six as A. ferridurans, YNTR4-15 Leptspirillum ferrooxidans and HBDY3-31 as Leptospirillum ferrodiazotrophum. The remaining strains were identified as A. ferrooxidans. Analysis of cbbL and cbbM genes sequences of representative 26 strains indicated that cbbL gene of 19 were two copies (cbbL1 and cbbL2) and 7 possessed only cbbL1. cbbM gene was single copy. In nucleotide-based trees, cbbL1 gene sequences of strains were separated into three sequence types, and the cbbL2 was similar to cbbL1 with three types. Codon bias of RubisCO genes was not obvious in Acidithiobacillus spp.. Conclusion: Strains isolated from three different regions in China indicated a great genetic diversity in Acidithiobacillus spp. and their 16S rRNA/RubisCO genes sequence was of significant difference. Phylogenetic tree based on 16S rRNA genes and RubisCO genes was different in Acidithiobacillus spp..

Characterization of the kinetics and electron paramagnetic resonance spectroscopic properties of Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR).

Acidithiobacillus ferrooxidans sulfide:quinone oxidoreductase (SQR) catalyzes the oxidation of sulfide to polysulfide chains or elemental sulfur coupled to quinone reduction via a non-covalent FAD cofactor. We investigated the role of the FAD using kinetics and EPR spectroscopy. The properties of the enzyme were compared with alanine and/or serine variants of conserved cysteine residues (Cys128, Cys160, Cys356) structurally close to the FAD cofactor and histidine residues (His132, His198) implicated in function. When the pre-steady state reduction of FAD was monitored, variants of Cys128 and His132 had similar rates to wild-type enzyme confirming they do not participate in the reductive half reaction whereas variants of Cys160, Cys356 and His198 had greatly reduced activity. Using steady state kinetics of Na2S-dependent decylubiquinone (DUQ) reduction we measured a kcat of 6.5s(-1) and a Km (Na2S) of 3.0µM and a Km (DUQ) of 3.4µM. Variants of Cys160, Cys356 and His198 had greatly diminished DUQ reduction activity whereas variants of Cys128 and His132 were less affected. A neutral flavin semiquinone was observed in the EPR spectrum of SQR reduced with Na2S which was enhanced in the Cys160Ala variant suggesting the presence of a Cys356-S(γ)-S-C(4A)-FAD adduct. Potentiometric titrations of the FAD semiquinone revealed an Em of -139±4mV at pH 7.0.

Identification and characterization of an ETHE1-like sulfur dioxygenase in extremely acidophilic Acidithiobacillus spp.

Elemental sulfur (S(0)) oxidation in Acidithiobacillus spp. is an important process in metal sulfide bioleaching. However, the gene that encodes the sulfur dioxygenase (SDO) for S(0) oxidation has remained unclarified in Acidithiobacillus spp. By BLASTP with the eukaryotic mitochondrial sulfur dioxygenases (ETHE1s), the putative sdo genes (AFE_0269 and ACAL_0790) were recovered from the genomes of Acidithiobacillus ferrooxidans ATCC 23270 and Acidithiobacillus caldus MTH-04. The purified recombinant proteins of AFE_0269 and ACAL_0790 exhibited remarkable SDO activity at optimal mildly alkaline pH by using the GSH-dependent in vitro assay. Then, a sdo knockout mutant and a sdo overexpression strain of A. ferrooxidans ATCC 23270 were constructed and characterized. By overexpressing sdo in A. ferrooxidans ATCC 23270, a significantly increased transcriptional level of sdo (91-fold) and a 2.5-fold increase in SDO activity were observed when S(0) was used as sole energy source. The sdo knockout mutant of A. ferrooxidans displayed a slightly reduced growth capacity in S(0)-medium compared with the wild type but still maintained high S(0)-oxidizing activity, suggesting that there is at least one other S(0)-oxidizing enzyme besides SDO in A. ferrooxidans ATCC 23270 cells. In addition, no obvious changes in transcriptional levels of selected genes related to sulfur oxidation was observed in response to the sdo overexpression or knockout in A. ferrooxidans when cultivated in S(0)-medium. All the results might suggest that SDO is involved in sulfide detoxification rather than bioenergetic S(0) oxidation in chemolithotrophic bacteria.

The sole cysteine residue (Cys301) of tetrathionate hydrolase from Acidithiobacillus ferrooxidans does not play a role in enzyme activity.

Cysteine residues are absolutely indispensable for the reactions of almost all enzymes involved in the dissimilatory oxidation pathways of reduced inorganic sulfur compounds. Tetrathionate hydrolase from the acidophilic iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans (Af-Tth) catalyzes tetrathionate hydrolysis to generate elemental sulfur, thiosulfate, and sulfate. Af-Tth is a key enzyme in the dissimilatory sulfur oxidation pathway in this bacterium. Only one cysteine residue (Cys301) has been identified in the deduced amino acid sequence of the Af-Tth gene. In order to clarify the role of the sole cysteine residue, a site-specific mutant enzyme (C301A) was generated. No difference was observed in the retention volumes of the wild-type and mutant Af-Tth enzymes by gel-filtration column chromatography, and surprisingly the enzyme activities measured in the cysteine-deficient and wild-type enzymes were the same. These results suggest that the sole cysteine residue (Cys301) in Af-Tth is involved in neither the tetrathionate hydrolysis reaction nor the subunit assembly. Af-Tth may thus have a novel cysteine-independent reaction mechanism.

A new cytoplasmic monoheme cytochrome c from Acidithiobacillus ferrooxidans involved in sulfur oxidation.

Acidithiobacillus ferrooxidans can obtain energy from the oxidation of various reduced inorganic sulfur compounds (RISCs, e.g., sulfur) and ferrous iron in bioleaching so has multiple branched respiratory pathways with a diverse range of electron transporters, especially cytochrome c proteins. A cytochrome c family gene, afe1130, which has never been reported before, was found by screening the whole genome of A. ferrooxidans. Here we report the differential gene transcription, bioinformatics analysis, and molecular modeling of the protein encoded by the afe1130 gene (AFE1130). The differential transcription of the target afe1130 gene versus the reference rrs gene in the A. ferrooxidans, respectively, on the culture conditions of sulfur and ferrous energy sources was performed through quantitative reverse transcription polymerase chain reaction (qRT-PCR) with a SYBR green-based assay according to the standard curves method. The qRT-PCR results showed that the afe1130 gene in sulfur culture condition was obviously more transcribed than that in ferrous culture condition. Bioinformatics analysis indicated that the AFE1130 was affiliated to the subclass ID of class I of cytochrome c and located in cytoplasm. Molecular modeling results exhibited that the AFE1130 protein consisted of 5 alpha-helices harboring one heme c group covalently bonded by Cys13 and Cys16 and ligated by His17 and Met62 and owned a big raised hydrophobic surface responsible for attaching to inner cytomembrane. So the AFE1130 in A. ferrooxidans plays a role in the RISCs oxidation in bioleaching in cytoplasm bound to inner membrane.

Tetrathionate-forming thiosulfate dehydrogenase from the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans.

Thiosulfate dehydrogenase is known to play a significant role in thiosulfate oxidation in the acidophilic, obligately chemolithoautotroph, Acidithiobacillus ferrooxidans. Enzyme activity measured using ferricyanide as the electron acceptor was detected in cell extracts of A. ferrooxidans ATCC 23270 grown on tetrathionate or sulfur, but no activity was detected in ferrous iron-grown cells. The enzyme was enriched 63-fold from cell extracts of tetrathionate-grown cells. Maximum enzyme activity (13.8 U mg(-1)) was observed at pH 2.5 and 70°C. The end product of the enzyme reaction was tetrathionate. The enzyme reduced neither ubiquinone nor horse heart cytochrome c, which serves as an electron acceptor. A major protein with a molecular mass of ∼25 kDa was detected in the partially purified preparation. Heme was not detected in the preparation, according to the results of spectroscopic analysis and heme staining. The open reading frame of AFE_0042 was identified by BLAST by using the N-terminal amino acid sequence of the protein. The gene was found within a region that was previously noted for sulfur metabolism-related gene clustering. The recombinant protein produced in Escherichia coli had a molecular mass of ∼25 kDa and showed thiosulfate dehydrogenase activity, with maximum enzyme activity (6.5 U mg(-1)) observed at pH 2.5 and 50°C.

Crystallization and preliminary X-ray diffraction analysis of tetrathionate hydrolase from Acidithiobacillus ferrooxidans.

Tetrathionate hydrolase (4THase) from the iron- and sulfur-oxidizing bacterium Acidithiobacillus ferrooxidans catalyses the disproportionate hydrolysis of tetrathionate to elemental sulfur, thiosulfate and sulfate. The gene encoding 4THase (Af-tth) was expressed as inclusion bodies in recombinant Escherichia coli. Recombinant Af-Tth was activated by refolding under acidic conditions and was then purified to homogeneity. The recombinant protein was crystallized in 20 mM glycine buffer pH 10 containing 50 mM sodium chloride and 33%(v/v) PEG 1000 using the hanging-drop vapour-diffusion method. The crystal was a hexagonal cylinder with dimensions of 0.2 × 0.05 × 0.05 mm. X-ray crystallographic analysis showed that the crystal diffracted to 2.15 Å resolution and belongs to space group P3(1) or P3(2), with unit-cell parameters a = b = 92.1, c = 232.6 Å.

HdrC2 from Acidithiobacillus ferrooxidans owns two iron-sulfur binding motifs but binds only one variable cluster between [4Fe-4S] and [3Fe-4S].

The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was suggested to own novel features that act in reverse to convert the sulfane sulfur of GS( n )H species (n > 1) into sulfite in sulfur oxidation. The HdrC subunit is potentially encoded by two different highly upregulated genes sharing only 29 % identity in A. ferrooxidans grown in sulfur-containing medium, which were named as HdrC1 and HdrC2, respectively and had been confirmed to contain iron-sulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe-4S] cluster by mutations. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here, we report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans. This HdrC2 had two identical motifs (Cx(2)Cx(2)Cx(3)C) containing total of eight cysteine residues potentially for iron-sulfur cluster binding. This purified HdrC2 was exhibited to contain one variable cluster converted between [4Fe-4S] and [3Fe-4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of these eight residues further confirmed that the HdrC2 in reduction with Fe(2+) condition loaded only one [4Fe-4S](+) with spin S = 1/2 ligated by the residues of Cys73, Cys109, Cys112, and Cys115; the HdrC2 in natural aeration condition lost the Fe atom ligated by the residue of Cys73 and loaded only one [3Fe-4S](0) with spin S = 0; the HdrC2 in oxidation condition loaded only one [3Fe-4S](+) with spin S = 1/2. Molecular modeling results were also in line with the experiment results.

Expression, purification and molecular modeling of another HdrC from Acidithiobacillus ferrooxidans which binds only one [4Fe-4S] cluster.

The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was predicted to have novel features that work in reverse to catalyse the sulfane sulfur of GSnH species (n > 1) into sulfite in sulfur oxidation. There are two different highly upregulated genes potentially encoding the HdrC subunit in A. ferrooxidans grown in sulfur-containing medium. An HdrC containing iron-sulfur cluster from A. ferrooxidans corresponding to one of the genes had been expressed and biophysically characterized. Comparatively, here we report the cloning, expression, and characterization of another HdrC from A. ferrooxidans. This HdrC was expressed in inclusion bodies in all conditions tested. This purified HdrC displayed brown color and contained the [4Fe-4S] cluster confirmed by the UV-scanning and EPR spectra. This HdrC owned two identical motifs (Cx(2)Cx(2)Cx(3)C) including total of eight cysteine residues for [4Fe-4S] cluster binding. To our surprise, the site-directed mutagenesis results of these eight residues revealed that respective removal of the sulfhydryl group of Cys73, Cys76, Cys79, and Cys37 resulted in the cluster loss, but those of Cys27, Cys30, Cys33, and Cys83 had no influence, which demonstrated that this HdrC bound only one cluster, and it might be responsible for causing the HdrABC in A. ferrooxidans working in reverse. Molecular modeling results also supported the above results and showed that this cluster was ligated by Cys73, Cys76, and Cys79 in one motif and Cys37, however, in another motif.

An extended ß7α7 substrate-binding loop is essential for efficient catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the biosynthetic pathway for the formation of 3-deoxy-D-manno-octulosonate, an important component in the cell wall of Gram-negative bacteria. KDO8P synthase is evolutionarily related to the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase, which uses erythrose 4-phosphate in place of A5P. The A5P binding site in KDO8P synthase is formed by three long loops that extend from the core catalytic (ß/α)(8) barrel, ß2α2, ß7α7, and ß8α8. The extended ß7α7 loop is always present in KDO8P synthase yet is not observed for DAH7P synthase. Modeling of this loop indicated interactions between this loop and the extended ß2α2 loop; both loops provide key hydrogen-bonding contacts with A5P. The two absolutely conserved residues on the ß7α7 loop (Gln and Ser) were mutated to Ala in both the metal-dependent KDO8P synthase from Acidithiobacillus ferrooxidans and the metal-independent KDO8P synthase from Neisseria meningitidis. In addition, mutants were constructed for both enzymes with the extended ß7α7 loop excised to match the DAH7P synthase architecture. Removal of the loop extension severely hindered efficient catalysis, dramatically increasing the K(m)(A5P) and reducing the k(cat) for both enzymes. Excision of the complete loop was far more detrimental to catalysis than the double mutations of the two conserved Gln and Ser residues. Therefore, the presence of the entire extended ß7α7 loop is important for efficient catalysis by KDO8P synthase, with the loop acting to promote efficient and productive binding of A5P.

An unconventional copper protein required for cytochrome c oxidase respiratory function under extreme acidic conditions.

Very little is known about the processes used by acidophile organisms to preserve stability and function of respiratory pathways. Here, we reveal a potential strategy of these organisms for protecting and keeping functional key enzymes under extreme conditions. Using Acidithiobacillus ferrooxidans, we have identified a protein belonging to a new cupredoxin subfamily, AcoP, for "acidophile CcO partner," which is required for the cytochrome c oxidase (CcO) function. We show that it is a multifunctional copper protein with at least two roles as follows: (i) as a chaperone-like protein involved in the protection of the Cu(A) center of the CcO complex and (ii) as a linker between the periplasmic cytochrome c and the inner membrane cytochrome c oxidase. It could represent an interesting model for investigating the multifunctionality of proteins known to be crucial in pathways of energy metabolism.

Specificity and mutational analysis of the metal-dependent 3-deoxy-D-manno-octulosonate 8-phosphate synthase from Acidithiobacillus ferrooxidans.

3-deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenol pyruvate and D-arabinose 5-phosphate to generate KDO8P. This reaction is part of the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Two distinct groups of KDO8PSs exist, differing by the absolute requirement of a divalent metal ion. In this study Acidithiobacillus ferrooxidans KDO8PS has been expressed and purified and shown to require a divalent metal ion, with Mn2+, Co2+ and Cd2+ (in decreasing order) being able to restore activity to metal-free enzyme. Cd2+ significantly enhanced the stability of the enzyme, raising the Tm by 14 degrees C. D-glucose 6-phosphate and D-erythrose 4-phosphate were not substrates for A. ferrooxidans KDO8PS, whereas 2-deoxy-D-ribose 5-phosphate was a poor substrate and there was negligible activity with D-ribose 5-phosphate. The 243AspGlyPro245 motif is absolutely conserved in the metal-independent group of synthases, but the Gly and Pro sites are variable in the metal-dependent enzymes. Substitution of the putative metal-binding Asp243 to Ala in A. ferrooxidans KDO8PS gave inactive enzyme, whereas substitutions Asp243Glu or Pro245Ala produced active enzymes with altered metal-dependency profiles. Prior studies indicated that exchange of a metal-binding Cys for Asn converts metal-dependent KDO8P synthase into a metal-independent form. Unexpectedly, this mutation in A. ferrooxidans KDO8P synthase (Cys21Asn) gave inactive enzyme. This finding, together with modest activity towards 2-deoxy-D-ribose 5-phosphate suggests similarities between the A. ferrooxidans KDO8PS and the related metal-dependent 3-deoxy-D-arabino-heptulosonate phosphate synthase, and highlights the importance of the AspGlyPro loop in positioning the substrate for effective catalysis in all KDO8P synthases.

Cellular levels of heme affect the activity of dimeric glutamyl-tRNA reductase.

Glutamyl-tRNA reductase (GluTR) is the first enzyme committed to tetrapyrrole biosynthesis by the C(5)-pathway. This enzyme transforms glutamyl-tRNA into glutamate-1-semi-aldehyde, which is then transformed into 5-amino levulinic acid by the glutamate-1-semi-aldehyde 2,1-aminomutase. Binding of heme to GluTR seems to be relevant to regulate the enzyme function. Recombinant GluTR from Acidithiobacillus ferrooxidans an acidophilic bacterium that participates in bioleaching of minerals was expressed in Escherichia coli and purified as a soluble protein containing type b heme. Upon control of the cellular content of heme in E. coli, GluTR with different levels of bound heme was obtained. An inverse correlation between the activity of the enzyme and the level of bound heme to GluTR suggested a control of the enzyme activity by heme. Heme bound preferentially to dimeric GluTR. An intact dimerization domain was essential for the enzyme to be fully active. We propose that the cellular levels of heme might regulate the activity of GluTR and ultimately its own biosynthesis.

Effects of copper exposure on expression of glutathione-related genes in Acidithiobacillus ferrooxidans.

The response of Acidithiobacillus ferrooxidans to variations in extracellular Cu exposure was investigated in terms of glutathione-related genes expression profiling based on reverse-transcription quantitative PCR analysis. The results show that the higher concentration of Cu would induce the expression of glutathione-related enzymes and cells elicited specific transcriptional responses when challenged with environmental Cu (0.08 mol l(-1)) conditions over a 60-min period. In comparison to the control, glutathione S-transferases (GST) and glutathione reductase (GR) were highly expressed when the cells were grown in the medium with copper, and the increase of glutathione and glutathione-related enzymes makes the cells acclimate to oxidative stress induced by Cu and protects the cells from toxicity caused by Cu exposure. It suggests that in order for Acidithiobacillus ferrooxidans to counteract the conditions of external Cu exposure, it modulated its expression level of GST, GR, glutathione hydrolase, and glutathione synthetase, which may protect organisms from oxidative damage. These parameters may be used to assess the biological impact of Cu in mining activities.