Abstract
Despite several discoveries in recent years, the physiology of acidophilic Micrarchaeota remains largely enigmatic. “Candidatus Micrarchaeum harzensis A_DKE”, for example, highly expresses numerous genes encoding hypothetical proteins and their function is difficult to elucidate due to a lacking genetic system. Still, not even the intracellular pH value of A_DKE is known, and heterologous production attempts are generally missing so far. Hence, A_DKE’s isocitrate dehydrogenase (MhIDH) was recombinantly produced in Escherichia coli and purified for bio-chemical characterisation. MhIDH appeared to be specific for NADP+, yet promiscuous regarding divalent cations as cofactors. Kinetic studies showed KM-values of 53.03±5.63 µM and 1.94±0.12 mM and kcat-values of 38.48±1.62 s-1 and 43.99±1.46 s-1 for DL-isocitrate and NADP+, respectively. MhIDH’s exceptionally low affinity for NADP+, potentially limiting its reaction rate, can be likely attributed to the presence of a proline residue in the NADP+ binding-pocket, which might cause a decrease in hydrogen bonding of the cofactor and a distortion of local secondary structure. Furthermore, a pH optimum of 7.89 implies, that A_DKE applies potent mechanisms of proton homoeostasis, to maintain a slightly alkaline cytosolic milieu in a highly acidic environment.
1. Introduction
Microorganisms can survive and thrive under extreme environmental conditions [1–3]. Bacteria and Archaea in particular are often adapted to niches of extreme temperature, pressure, radiation, salinity or pH, which allows them to populate a vast variety of habitats inaccessible to non-extremophiles [1,4]. Still, to cope with these conditions, requires a significant amount of metabolic resources, in order to adjust the intracellular reaction conditions.
Acidophilic Archaea, for example, are thriving in environments with pH values below pH 3 [5,6], in extreme cases, optimal growth occurs close to pH 0 (i.e. Picrophilus torridus [7], Ferroplasma acidiphilum [8]). Still, these organisms are able to maintain less acidic to near neutral internal pH (pHi) values between pH 4.6 (i.e. Picrophilus torridus, [9]) and pH 6.5 (i.e. Sulfolobus acidocaldarius, [10]) by applying numerous synergistic mechanisms of proton homoeostasis [5,11,12].
Micrarchaeota were originally discovered in habitats with pH values between 0.5 and 4.0 [13]. Common characteristics of known members of this phylum are small-sized, circular genomes and an overall limited metabolic potential [13–17]. Thus, Micrarchaeota are assumed to be dependent on a symbiotic relationship with host organisms of the order Thermoplasmatales [15,18,19].
To our best knowledge, the only acidophilic Micrarchaeon currently cultivated under laboratory conditions is “Candidatus Micrarchaeum harzensis A_DKE” in co-culture with its putative host “Ca. Scheffleriplasma hospitalis B_DKE” [17]. The culture was enriched from acid mine drainage biofilms originating from the abandoned pyrite mine “Drei Kronen und Ehrt” in the Harz Mountains (Germany) [18,20]. Optimal growth of the laboratory culture was achieved at pH 2 [17]. Although an extensive multi-omic-approach, comprising genomics, transcriptomics, proteomics, and metabolomics, has been conducted on A_DKE [17,21], details of its metabolism still remain enigmatic. Approximately a third of the genes in the A_DKE genome encode hypothetical proteins, most of which are also actively expressed, according to transcriptomic data [17]. Of note, these hypothetical protein-encoding genes comprise 35 % and 60 % of A_DKE’s 100 and 10 highest expressed genes, respectively [unpublished data]. Considering A_DKEs reduced genome and so far largely enigmatic metabolism [17,18], these proteins of unknown function might be crucial for understanding A_DKE’s physiology. Yet, due to low sequence conservation, in silico characterisation of these proteins is currently not possible and thus biochemical characterisation remains key to fully understand A_DKE’s physiology. Investigating the function of these proteins by means of heterologous expression proves to be difficult, since there is no information on the intracellular conditions in Micrarchaeota. One of the factors defining the intracellular conditions and protein stability of an organism is the pHi value, as it affects the activity of proteins, for example in DNA transcription, protein synthesis and biocatalysis (for reviews, please check [11,22]). Thus, a suitable production platform must be chosen mimicking the intracellular conditions of A_DKE as best as possible to facilitate proper folding of the proteins of interest.
The goal of this study was to gain evidence for the pHi of A_DKE by biochemical characterisation of an intracellular enzyme. As a target protein, its isocitrate dehydrogenase (IDH) was chosen, which is a key enzyme of the tricarboxylic acid cycle catalysing the oxidative decarboxylation of isocitrate to α-ketoglutarate and CO2 [23]. This analysis revealed a slightly alkaline pH optimum indicating that A_DKE displays a comparatively high pHi for an acidophile.
2. Materials and Methods
2.1 Database Research and Bioinformatic Sequence and Structure Analyses
Genomic (accession number: CP060530) and transcriptomic data (accession numbers: SRX8933312-SRX8933315) of A_DKE were accessed via the National Center for Biotechnology Information NCBI [24] (bio project number: PRJNA639692). The pH optima and kinetic parameters of homologous enzymes for comparison with experimentally identified parameters for MhIDH were obtained from the BRENDA database ([25], www.brenda-enzymes.org).
The theoretical molecular weight and isoelectric point of MhIDH were calculated using the CLC Main Workbench 20.0.1 (QIAGEN, Aarhus, Denmark). Conserved sequence motifs and protein domains were detected using the Pfam database ([26], www.pfam.xfam.org). MhIDH homologues were identified via BLASTp [27] search of the UniprotKB/swiss-prot database [24] via NCBI. A multiple sequence alignment comparing MhIDH with experimentally verified homologues from Escherichia coli K-12 (EcIDH, NCBI: P08200.1), Aeropyrum pernix K1 (ApIDH, NCBI: GBF08417.1), Archaeoglobus fulgidus DSM 4304 (AfIDH, NCBI: O29610.1), Haloferax volcanii DS2 (HvIDH, NCBI: D4GU92.1) and Sulfolobus tokodaii Strain 7 (StIDH, NCBI: BAB67271.1) was carried out using the Clustal Omega algorithm [28–30] as a plugin for the CLC Main Workbench 20.0.1. The alignment was visualised using the ESPript 3.0 server ([31], www.espript.ibcp.fr).
Homology modelling of a putative MhIDH structure was achieved via the CLC Main Workbench 20.0.1 using the crystal structure of EcIDH in complex with Ca2+, isocitric acid and NADP+ ([32], PDB: 4AJ3, 49.5 % homology, 1.9 Å resolution) as a template. Assessment of local model quality and B-factor, as well as docking of the cofactors Mn2+, NADP+ and the substrate isocitrate to the MhIDH model structure was performed using the ResQ server [33] and the COACH server [34,35] respectively. Protein ligand interactions were analysed using the PLIP server ([36], www.plip-tool.biotec.tu-dresden.de/plip-web). All protein structures were visualised using PyMOL 2.3.3 (Schrödinger, Ney York, USA).
2.2 Cloning and Recombinant Expression of icd26x His
The icd2 gene was PCR-amplified from genomic DNA isolated from a co-culture containing “Ca. Micrarchaeum harzensis A_DKE” and “Ca. Scheffleriplasma hospitalis B_DKE” [17] via oligonucleotide primers 1 & 2 (see Table 1). The latter introduced a 6x His-tag encoding sequence to the 5’-end, as well as complementary overlaps to the target vector pBAD202 (Invitrogen, Carlsbad, CA, USA). pBAD202 was linearised via inverse PCR using primers 3 & 4 (see Table 1). Both PCR products were gel-purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Mannheim, Germany) and assembled via isothermal in vitro ligation [37]. The resulting plasmid pBAD202_icd26x His was transformed into E. coli Rosetta pRARE (Merck, Darmstadt, Germany).
In order to monitor production of MhIDH6x His over time, E. coli Rosetta pRARE pBAD202_icd26x His was cultivated in shaking flasks containing 50 mL Terrific Broth medium (1.2 % (w/v) tryptone, 2.4 % (w/v) yeast extract, 0.5 % (w/v) glycerol, 17 mM KH2PO4, 72 mM K2HPO4) supplemented with 50 µg mL-1 kanamycin and 30 µg mL-1 chloramphenicol at 37 °C and 180 rpm. Upon reaching an OD600 of 0.6-0.8, expression of icd26x His was induced by addition of 1 mM L-(+)-arabinose. From this point forth, the culture was incubated at 30 °C and 180 rpm and samples (1 mL) were taken at different time points after induction (0, 1, 2, 4, 6 and 24 h), and subjected to OD600-measurement using a GENESYSTM 20 spectrophotometer (Thermo Fisher Scientific, Schwerte, Germany) and preparation for SDS-PAGE analysis. Samples were centrifuged for 2 min at 16 000 g and cell pellets were resuspended in 75 µL of 2x SDS loading dye (240 mM TRIS/HCl (pH 6.8), 20 % (v/v) glycerol, 2 % (w/v) SDS, 100 mM DTT, 0.02 % (w/v) Orange G) per OD600 of 0.2, boiled for 10 min at 95 °C and centrifuged for 5 min at 16 000 g. After determination of the optimal induction time, over-expression was carried out in a total volume of 1 L as described above. Cells were harvested for 15 min at 16 000 g and 4 °C, 4 h after induction and stored at 20 °C until used.
2.3 Isolation and Affinity Purification of MhIDH6x His
The cell pellet of an expression culture was resuspended in IMAC buffer (50 mM HEPES/NaOH (pH 7.4), 500 mM NaCl) followed by the addition of a spatula tip of Deoxyribonuclease I (SERVA Electrophoresis, Heidelberg, Germany). Cell extracts were prepared using mechanical disruption in an FA-078 FRENCH® Pressure Cell Press (SLM Aminco, Urbana, IL, USA) at 137.8 MPa. The raw lysate was fractioned by suc-cessive steps of centrifugation. Intact cells and cell debris were pelleted for 15 min at 6 000 g and 4 °C. Membranes were separated from the plasma fraction via ultracentrifugation for 60 min at 138 000 g and 4 °C. The membrane pellet was resuspended in solubilisation buffer (20 mM HEPES/NaOH (pH 8.0), 150 mM NaCl, 2 % (v/v) Triton X-100) and the plasma fraction was passed through a 0.2 µm syringe filter (Sarstedt, Nümbrecht, Germany) to remove remaining insoluble particles. Samples of the raw lysate, as well as the membrane and plasma fraction were used for SDS-PAGE.
Nickel Immobilised Metal Ion Affinity chromatography (Ni2+-IMAC) for protein purification was conducted using a HisTrap® HP 5 mL column (GE Healthcare, Munich, Germany) coupled to a BioLogic DuoFlow™ Chromatography System (Bio-Rad, Munich, Germany). The column was equilibrated with IMAC buffer, prior to loading with plasma fraction. Non-specifically bound proteins were removed by washing with IMAC buffer containing 80 mM imidazole. Elution of the target protein was achieved with IMAC buffer containing 500 mM imidazole. The eluted fraction was concentrated using a 3 kDa MWCO centrifugal filter (Merck, Darmstadt, Germany). Samples of the column flow-through, wash and eluate were used for SDS-PAGE.
Size exclusion chromatography (SEC) of the concentrated protein solution was conducted using a HiLoad™ 26/600 Superdex™ 200 pg column (GE Healthcare, Mu-nich, Germany) coupled to the aforementioned chromatography system. The column was equilibrated and run isocratically with IDH buffer (50 mM HEPES/NaOH (pH 7.4), 150 mM NaCl, 1 mM DTT, 0.5 mM MgCl2). The eluted fractions were collected, concentrated and analysed via SDS-PAGE. For long term storage at -20 °C, 50 % (v/v) glycerol was added.
2.4 Protein Quantification, SDS-PAGE & Western Blot
Protein quantification of samples collected for analysis via SDS-PAGE was carried out according to [38]. Alternatively, purified protein was quantified spectrophotometrically using a NanoDrop 2000 (Thermo Fisher Scientific, Schwerte, Germany).
Samples containing 20 µg of total protein (5 µg in case of purified protein) were mixed with 2x SDS loading dye and separated via denaturing SDS-PAGE in hand cast 12 % TRIS/Glycine gels according to [39]. As reference either BlueStar™ Prestained Protein Marker (NIPPON Genetics, Düren, Germany) or PageRuler™ Prestained Protein Ladder (Thermo Fisher Scientific, Schwerte, Germany) was used. After separation, the gels were subjected to either colloidal staining using Quick Coomassie Stain (Protein Ark, Sheffield, UK) or transfer of the separated proteins to a nitrocellulose membrane (Roth, Karlsruhe, Germany) via a semi-dry blot. The latter was carried out with a Trans-Blot® Turbo™ device (Bio-Rad, Munich, Germany) at 1.3 A for 10 min using a continuous blotting buffer system (330 mM TRIS, 267 mM glycine, 15 % (v/v) ethanol, 5 % (v/v) methanol, pH 8.8).
Densitometric estimation of protein purity from Coomassie-stained acrylamide gels was carried out using the Image Studio Lite 5.2 software (LI-COR, Lincoln, NE, USA).
For immuno-staining the membrane was blocked for at least 1 h at room temperature with TBST (20 mM TRIS/HCl (pH 7.5), 500 mM NaCl, 0.05 % (v/v) Tween® 20) containing 3 % (w/v) skim milk powder. After a few brief rinses with TBST, the blot was incubated with a mouse anti-His-tag primary antibody (Sigma-Aldrich, Steinheim, Germany), diluted 1:1 000 in TBS (10 mM TRIS/HCl (pH 7.5), 150 mM NaCl) containing 3 % (w/v) BSA for 1 h, followed by washing with TBST (4x 5 min) and incubation with a goat anti-mouse alkaline phosphatase secondary antibody (Sigma-Aldrich, Steinheim, Germany) diluted 1:30 000 in TBST containing 3 % (w/v) skim milk powder for 45 min. After washing with TBST (4x 5 min) and several brief rinses with dH2O, protein bands were visualised colorimetrically using the AP conjugate substrate kit (Bio-Rad, Munich, Germany) according to manufacturer’s instructions.
2.5 Spectrophotometric IDH-Activity Assays and Determination of Kinetic Properties
MhIDH6x His activity and kinetic properties were determined at least in triplicates at 28 °C by monitoring the formation of NADH or NADPH spectrophotometrically at 340 nm using an NADH or NADPH standard curve for quantification. The standard reaction mixture contained 100 mM TRIS/HCl (pH 8.0), 1 mM DL-Na3-isocitrate, 5 mM MgCl2, 2 mM Na2NADP and 0.6-2.5 µg enzyme in a total volume of 200 µL. Each reaction was started individually by addition of either NADP+ or enzyme using a TeInjectTM Dispenser (Tecan, Männedorf, Swiss) followed by measurement of A340 each 200 ms for 15-30 s using an Infinite® M 200 PRO plate reader (Tecan, Männedorf, Swiss). Investigation of cofactor-specificity was conducted by measuring specific activity with 20 mM NADP+ or NAD+ in presence of Mg2+ and cation-dependency was determined by measuring specific activity in presence of 5 mM MgCl2, MnCl2, CaCl2, ZnCl2, NiCl2, CuCl2, CoCl2 and Na2EDTA, respectively, with 2 mM NADP+. The pH optimum was determined by measuring specific activity in buffers with varying pH values. A corresponding polynomial fitting curve of 5th order was calculated using Origin Pro 2020. In order to span a range from pH 5 to 9.5, three different buffer systems were applied as described in [40]: 0.1 M CH3CO2Na/CH3CO2H (pH 5.0-6.0), 0.1 M Na2HPO4/NaH2PO4 (pH 5.5-7.5), and 0.1 M TRIS/HCl (pH 7.0-9.5). Enzyme kinetics were determined by measuring the initial reaction rate at increasing concentrations of NADP+ (0-10 mM) and DL-isocitrate (0-500 µM), respectively. KM and Vmax were calcu-lated from a non-linear fit based on the Michaelis-Menten model [41,42] using Origin Pro 2020.
3. Results and Discussion
3.1 MhIDH Shows Conserved Characteristics of Prokaryotic, NADP-Dependent IDHs
pHi values can be estimated from the pH optima of cytosolic enzymes [43]. The enzyme of choice should be monomeric or homo-oligomeric and preferably allow cost-effective and direct activity measurement. The isocitrate dehydrogenase (IDH) of A_DKE fulfils these requirements.
A_DKE possesses only one gene (icd2, Micr_00902) annotated to be encoding a putative NADP-dependent IDH, which is actively expressed, according to available transcriptomic data [17]. In silico analyses of its amino acid sequence allowed the calculation of a theoretical molecular weight and isoelectric point (pI) of 45.05 kDa and 5.82, respectively, as well as the discovery of a highly conserved isocitrate/isopropylmalate dehydrogenase domain (Pfam: PF00180.20), almost spanning the entire length of the sequence (Thr23-Leu402). Furthermore, a BLASTp search of the UniprotKB/swiss-prot database revealed high sequence homology to several experimentally proven homo-dimeric, NADP-dependent IDHs with nearly all amino acids reported to be involved in substrate and cofactor binding being conserved (see Figure A1 and Table A1). Hence, this bioinformatic data strongly suggests that this protein is indeed an IDH and thus can be used for biochemical characterisation.
3.2. MhIDH6x His Can be Produced in E. coli
Since direct purification of native MhIDH from “Ca. Micrarchaeum harzensis A_DKE” is not feasible due to only low cell density cultures, the corresponding gene was cloned and over-expressed in E. coli. Test-expression over time showed high ex-pression levels with a maximum at 4 h after induction and no significant degradation of the product, even 24 h after induction (see Figure 1a). The protein has an apparent molecular weight of roughly 50 kDa, matching the theoretical molecular weight. It was found to be located in the cytoplasmic fraction and could not be detected in the membrane fraction (see Figure 1b). Affinity purification of MhIDH6x His from the plasma fraction was successful in a single step, providing roughly 90 % of electrophoretic homogeneity (see Figure 1c). SEC was used for further purification.
3.3 Biochemical Properties of MhIDH6x His
3.3.1 MhIDH6x His Activity is Dependent on NADP+ and Divalent Cations
IDHs catalyse the oxidative decarboxylation of isocitrate to α-ketoglutarate and CO2. The electrons released in this process are transferred to either NAD+ (EC 1.1.1.41) or NADP+ (EC1.1.1.42) [23,44]. Type I IDHs found in Bacteria and Archaea predominantly use NADP+ [44,45]. Still, promiscuous forms accepting both cofactors have been reported as well [46–48]. Furthermore, IDHs are known to be dependent on divalent metal cations, such as Mg2+ and Mn2+ [49]. In order to characterise enzyme activity of recombinant MhIDH, its dependency on different cofactors was tested.
With 41.09±1.02 µmol min-1 mg-1 MhIDH6x His activity is about 55-fold higher using NADP+ as cofactor relative to NAD+ with only 0.74±0.09 µmol min-1 mg-1 (see Figure 2a). The apparent NADP+ specificity of the enzyme is also supported by structural data. The primary structure of MhIDH contains conserved amino acid residues (Lys335, Tyr336 and Arg386) in the active site (see Figure A1), which have been shown in EcIDH [50,51], StIDH [52] and ApIDH [53] to specifically stabilise the 2’-phosphate moiety of NADP+ ensuring that NADP+ is bound preferably.
As expected, divalent cations appear to be vital for MhIDH6x His function, as the enzyme does not show any activity in presence of EDTA (see Figure 2b). Still, with several different metal ions having an activating effect, MhIDH is rather promiscuous in this regard. While Mn2+ and Mg2+ induced maximal activity increases, only 44.2±4.01 %, 43.2±1.99 % and 6.6±0.60 % of relative maximal activity can be achieved with Cu2+, Co2+, and Ni2+, respectively. Zn2+ and Ca2+, on the other hand, do not seem to enhance enzyme activity, as in presence of these ions MhIDH6x His is only marginally more active than in presence of EDTA. The variance in activation levels in presence of different cations is seemingly independent of ionic radii and is hypothesised to be due to individual modes of binding in the active site of the enzyme [54]. Moreover, Zn2+ [55] and Ca2+ [54,56] have been reported to inhibit IDH activity. In case of Ca2+, this is most likely due to a spatial shift of ligands bound in the active site in order to accommodate the large ionic radius of the cation [56].
3.3.2 MhIDH6x His Shows Highest Activity at Slightly Alkaline pH
With the optimal cofactor combination known, specific activity was measured at different pH values in increments of 0.5. From this data a non-linear fitting curve was calculated with the global maximum of the curve indicating the pH optimum of the enzyme, which was identified to be pH 7.89. At least 90 % of the maximum specific activity could be retained in a range from pH 7.39 to 8.35 (see Figure 3a). A comparison to other IDHs, listed in the BRENDA database reveals this feature to be quite common, as it is close to the median value of pH 8 (see Figure 3b).
Note however, that IDHs in this comparison exclusively originate from neutralophilic organisms, since to our knowledge data on pH optima of IDHs from acidophilic organisms is scarce. One of these few cases being Thermoplasma acidophilum IDH (TaIDH). Growing optimally in environments with pH values of 1-2, T. acidophilum has a pHi value of 5.8 [57]. Contrary to that, TaIDH displays optimal activity at pH 7.5 [58]. Still, TaIDH is reported to retain a third of its maximal specific activity at pH 5.8 [58], which is not the case for MhIDH6x His. Moreover, other enzymes of acidophiles are reported to display highest activity at slightly acidic pH values [6,59,60]. This finding implies a higher intracellular pH of A_DKE compared to other acidophiles.
3.3.3 MhIDH6x His is Characterised by Low NADP+ Affinity
Kinetic data of MhIDH6x His was obtained for the substrate DL-isocitrate and the cofactor NADP+ (see Figure 4a & b). Overall, kinetic properties of MhIDH6x His regarding DL-isocitrate appear to be quite average compared to other IDHs (see Figure 4c and Table A2), as with KM = 53.03±5.63 µM, kcat = 38.48±1.62 s-1 and kcat/KM = 725±107.62 mM-1 s-1 all parameters lie close to the respective median value. Regarding NADP+, on the other hand, MhIDH6x His performs significantly worse in comparison to other IDHs (see Figure 4d and Table A2). A KM of 1.94±0.12 mM is exceptionally high compared to other IDHs being the least specific enzyme in the comparison. Despite a decent turnover rate close to the median value (kcat = 43.99±1.46 s-1), MhIDH6x His ranks among the three IDHs with the lowest catalytic efficiency (kcat/KM = 22.69±2.15 mM-1 s-1). All in all, low affinity to NADP+ seems to be the bottleneck limiting the overall reaction rate of MhIDH6x His and possibly the metabolic rate of the whole organism, given that IDH is a key enzyme of the tricarboxylic acid cycle, which is the central metabolic pathway in A_DKE [18].
To investigate potential ligand binding mechanisms in MhIDH, we conducted a multiple sequence alignment with other experimentally verified IDHs and modelled a putative structure (see Figure 5) using a crystal structure of EcIDH as a template. The model features high estimated local model quality and shows the characteristic fold of prokaryotic NADP-dependent IDHs, comprising a large and a small domain responsible for cofactor and substrate binding, respectively, as well as a clasp domain allow-ing homo-dimerisation ([52,53], see Figure 5b). The estimated local B-factor of the model indicates a rigid core, as well as flexible loops surrounding the active site in between the small and large domain (see Figure 5b), which allow conformational change necessary for catalytic activity in EcIDH [32]. Ligands isocitrate, NADP+ and Mn2+ could be docked in the active sites of the homo-dimeric model, with their relative positions closely resembling those in EcIDH (see Figure 5c).
A comparably average KM value for isocitrate is not surprising, considering that without exception all amino acids known to be involved in isocitrate binding in other IDHs [32,52,53,56] are conserved in the isocitrate binding pocket of MhIDH (see Figure 5a).
Furthermore, low affinity of MhIDH for NADP+ can be explained by structural analysis, as well. The NADP+ binding pocket in EcIDH is formed by the 310-helix η4 (residues 318-324), the NADP+ binding loop (residues 336-352), as well as helix α12 (residues 390-397) [32]. In particular, amino acids Lys100, Leu103, Thr105, Asn232*, amino acids 258*-261*, Trp263*, Gln287*, Gln288*, Arg292*, Glu336, His339, Gly340, Ala342, Lys344, Tyr345, Asn352, Tyr391 und Arg395 (* marks amino acids from the second subunit of the homo-dimer) are involved in binding NADP+ via hydrogen bonds or salt bridges ([32], see Figure 5a). Corresponding residues in StIDH [52] and ApIDH [53] have been described to facilitate NADP+ binding, as well (see Figure 5a). Almost all of the corresponding amino acids in MhIDH are conserved or at least dis-play similar physicochemical properties (Tyr254* instead of Trp263* and Lys282* in-stead of Arg292*), the only exception being Tyr391 (see Figure 5a & d), which is substituted for a proline in MhIDH (Pro382). While this appears to be a common feature among isopropylmalate dehydrogenases rather than IDHs (i.e. in Thermus thermophilus [61]), MhIDH showed significantly higher sequence homology to the latter (see Table A1). Since Tyr391 forms hydrogen bonds stabilising the 2’-phosphate of NADP+ (see Figure 5a & d) this amino acid plays a critical role in cofactor stabilisation and selectivity in EcIDH [50,51,61]. Moreover, it has been reported that a proline at this position disrupts the local α-helix in favour of a β-turn [61,62], which could distance Lys386, another crucial residue ensuring NADP+ specificity, from the 2’-phosphate of NADP+ and thereby decrease cofactor stabilisation even more.
4. Conclusion
Although several approaches lead to new findings about Micrarchaeota in the last decade, the survival strategies of these ultra-small, acidophilic organisms are still not fully understood. In this study, we gained evidence for the internal pH of “Ca. Micrarchaeum harzensis A_DKE”, by characterisation of its IDH. The enzyme was successfully produced in E. coli and biochemically characterised. Compared to other known IDHs, the NADP+ and divalent cation-dependent protein from A_DKE seems to be highly inefficient because of the amino acid composition of its NADP+ binding-pocket. Since MhIDH plays a role in A_DKE’s main pathway for generation of reducing equivalents, its inefficiency is in line with the slow growth rates of the Micrarchaeon.
Over the years, a vast arsenal of methods, suitable for the determination of pHi values has been developed, including cell homogenate measurement, pH-sensitive fluorescent proteins and fluorescent probes, injection of microelectrodes and 31P-NMR spectroscopy. All these methods come with individual strengths and weaknesses, discussed elsewhere [11,63]. In our specific case, however, experimental determination of the pH optimum of an intracellular enzyme as described in [6,43] remained the only viable option.
The presented data suggests that A_DKE maintains a slightly alkaline cytosolic milieu close to pH 8, while thriving in acidic environments of pH 2, resulting in a steep pH gradient of several orders of magnitude. Should this assumption be correct, A_DKE would have the highest pHi among all acidophiles described so far, which raises the question how the Micrarchaeon is able to maintain this pH gradient. In literature there are several synergistic strategies of proton homoeostasis described for acidophiles [5,11], many of which might apply to A_DKE as well:
Membranes consisting of archaeal tetraetherlipids have been reported to be highly impermeable for protons [5,64–67]. With a caldarchaeol content of 97 % the cell membrane of A_DKE is predominantly composed of such lipids [17]. Furthermore, in T. acidophilum HO-62 a correlation between acid tolerance and elevated levels of surface glycosylation has been found [68]. The cell surface of A_DKE is mostly covered by a proteinaceous, heavily glycosylated S-layer [21]. Since, biomimetic experiments strongly suggest, that polysaccharide chains attached to the cell surface might effectively create a proton shelter [69], the glycans linked to A_DKE’s S-layer, could allow further shielding from protons. Another mechanism of acidophiles to repel invading protons is the formation of a positive potential at the inside of the cell membrane via cation transporters [5,11,12,67]. Also, acidophiles are known to express a variety of primary proton transporters in order to counteract cellular protonation caused by ATP synthase activity [5,11,12]. According to transcriptomic data, A_DKE seems to express several genes encoding (putative) proton pumps and cation transporters (see Table A3), which would allow export of protons to the extracellular space, as well as antiport exchanging protons for cations.
Lastly, this study proves the viability of recombinant production of functional A_DKE proteins in E. coli, which opens numerous possibilities for the biochemical characterisation of proteins of unknown function in A_DKE.
Author Contributions
Conceptualisation, J.G.; methodology, D.W.; validation, D.W. and J.G.; formal analysis, D.W.; investigation, D.W.; resources, J.G.; data curation, D.W. and S.G.; writing—original draft preparation, D.W., S.G. and J.G.; writing—review and editing, D.W., S.G. and J.G.; visualisation, D.W.; supervision, J.G.; project administration, J.G.; funding acquisition, J.G. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Data Availability Statement
All data shown is contained within the article.
Conflicts of Interest
The authors declare no conflict of interest.
Appendix A
Footnotes
dennis.winkler{at}student.kit.edu; sabrina.gfrerer{at}kit.edu