Computer-aided rational design strategy based on protein surface charge to improve the thermal stability of a novel esterase from Geobacillus jurassicus.

OBJECTIVES: Although Geobacillus are significant thermophilic bacteria source, there are no reports of thermostable esterase gene in Geobacillus jurassicus or rational design strategies to increase the thermal stability of esterases. RESULTS: Gene gju768 showed a highest similarity of 15.20% to esterases from Geobacillus sp. with detail enzymatic properties. Using a combination of Gibbs Unfolding Free Energy (∆∆G) calculator and the distance from the mutation site to the catalytic site (DsCα-Cα) to screen suitable mutation sites with elimination of negative surface charge, the mutants (D24N, E221Q, and E253Q) displayed stable mutants with higher thermal stability than the wild-type (WT). Mutant E253Q exhibited the best thermal stability, with a half-life (T1/2) at 65 °C of 32.4 min, which was 1.8-fold of the WT (17.9 min). CONCLUSION: Cloning of gene gju768 and rational design based on surface charge engineering contributed to the identification of thermostable esterase from Geobacillus sp. and the exploration of evolutionary strategies for thermal stability.

High-resolution neutron crystallography visualizes an OH-bound resting state of a copper-containing nitrite reductase.

Copper-containing nitrite reductases (CuNIRs) transform nitrite to gaseous nitric oxide, which is a key process in the global nitrogen cycle. The catalytic mechanism has been extensively studied to ultimately achieve rational control of this important geobiochemical reaction. However, accumulated structural biology data show discrepancies with spectroscopic and computational studies; hence, the reaction mechanism is still controversial. In particular, the details of the proton transfer involved in it are largely unknown. This situation arises from the failure of determining positions of hydrogen atoms and protons, which play essential roles at the catalytic site of CuNIRs, even with atomic resolution X-ray crystallography. Here, we determined the 1.50 Šresolution neutron structure of a CuNIR from Geobacillus thermodenitrificans (trimer molecular mass of ∼106 kDa) in its resting state at low pH. Our neutron structure reveals the protonation states of catalytic residues (deprotonated aspartate and protonated histidine), thus providing insights into the catalytic mechanism. We found that a hydroxide ion can exist as a ligand to the catalytic Cu atom in the resting state even at a low pH. This OH-bound Cu site is unexpected from previously given X-ray structures but consistent with a reaction intermediate suggested by computational chemistry. Furthermore, the hydrogen-deuterium exchange ratio in our neutron structure suggests that the intramolecular electron transfer pathway has a hydrogen-bond jump, which is proposed by quantum chemistry. Our study can seamlessly link the structural biology to the computational chemistry of CuNIRs, boosting our understanding of the enzymes at the atomic and electronic levels.

Functional Studies of α-Riboside Activation by the α-Ribazole Kinase (CblS) from Geobacillus kaustophilus.

We report the initial characterization of the α-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus (GkCblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of cobamides. We implemented a continuous spectrophotometric assay to obtain kinetic parameters for several potential substrates and to study the specificity of the enzyme for α-N-linked ribosides. The apparent Km values for α-R and ATP were 358 and 297 µM, respectively. We also report methods for synthesizing and quantifying non-commercially available α-ribosides and ß-ribazole (ß-R). Purified GkCblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). GkCblS did not phosphorylate ß-N-linked glycosides like ß-adenosine or ß-R. Expression of G. kaustophilus cblS+ in a Salmonella enterica subsp. enterica sv Typhimurium LT2 (S. enterica) strain lacking the nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyl transferase (CobT) enzyme resulted in the activation of various benzimidazole α-ribosides, and the synthesis of benzimidazolyl cobamides to levels that supported robust growth. Notably, α-Ado did not support growth under similar conditions, in spite of the fact that GkCblS phosphorylated α-Ado in vitro. When α-Ado was provided at a very high concentration, growth was observed. This result suggested that in S. enterica α-Ado transport may be inefficient. We conclude that GkCblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.

Structural and functional characterization of a putative carbonic anhydrase from Geobacillus kaustophilus reveals its cambialistic function.

Carbonic anhydrases (CA) are the most ubiquitous ancient zinc metalloenzymes known. Here we report the structural and functional analysis of a hypothetical protein GK2848 from Geobacillus kaustophilus. The analysis revealed that it belongs to the γ-class of CA (termed as Cag). Only a limited number of γ-class CA's have been characterized till date. Interestingly Cag contains magnesium at its active site instead of a traditional zinc ion. Based on the structural and sequence comparison with similar γ-CA's the putative active site residues of Cag were identified. This analysis revealed that an important catalytic residue and a proton shuttle residue (Glu62 and Glu84 respectively) of Cam (previously characterized γ-CA from Methanosarcina thermophila) are absent in Cag, however certain other active site residues are conserved both in Cag and Cam. This suggests that Cag uses a different set of residues for the reversible hydration of CO2 to HCO3- when compared with Cam. Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES) and 25Mg and 67Zn NMR studies on Cag and its mutants revealed that either Mg or Zn can occupy the active site which suggests the cambialistic nature of the enzyme.

High-Throughput Solid-Phase Assay for Substrate Profiling and Directed Evolution of Transketolase.

Thiamine diphosphate-dependent enzymes, and specifically transketolases, form one of the most important families of biocatalytic tools for enantioselective carbon-carbon bond formation yielding various hydroxyketones of biological interest. To enable substrate profiling of transketolases for acceptance of different donors and acceptors, a simple, direct colorimetric assay based on pH reaction variation was developed to establish a high-throughput solid-phase assay. This assay reduces the screening effort in the directed evolution of transketolases, as only active variants are selected for further analysis. Transketolase activity is detected as bicarbonate anions released from the α-ketoacid donor substrate, which causes the pH to rise. A pH indicator, bromothymol blue, which changes color from yellow to blue in alkaline conditions, was used to directly detect, with the naked eye, clones expressing active transketolase variants, obviating enzyme extraction.

Pd-Oxazolone complexes conjugated to an engineered enzyme: improving fluorescence and catalytic properties.

Different Pd-complexes containing orthometallated push-pull oxazolones were inserted by supramolecular Pd-amino acid coordination on two genetically engineered modified variants of the thermoalkalophilic Geobacillus thermocatenolatus lipase (GTL). Pd-lipase conjugation was performed on the solid phase in the previously immobilized form of GTL under mild conditions, and soluble conjugated Pd-GTL complexes were obtained by simply desorbing by washing with an acetonitrile aqueous solution. Three different Pd complexes were incorporated into two different genetically modified enzyme variants, one containing all the natural cysteine residues changed to serine residues, and another variant including an additional Cys mutation directly in the catalytic serine (Ser114Cys). The new Pd-enzyme conjugates were fluorescent even at ppm concentrations, while under the same conditions free Pd complexes did not show fluorescence at all. The Pd conjugation with the enzyme extremely increases the catalytic profile of the corresponding Pd complex from 200 to almost 1000-fold in the hydrogenation of arenes in aqueous media, achieving in the case of GTL conjugated with orthopalladated 4a an outstanding TOF value of 27 428 min-1. Also the applicability of GTL-C114 conjugated with orthopalladated 4b in a site-selective C-H activation reaction under mild conditions has been demonstrated. Therefore, the Pd incorporation into the enzyme produces a highly stable conjugate, and improves remarkably the catalytic activity and selectivity, as well as the fluorescence intensity, of the Pd complexes.

The use of response surface methodology for enhanced production of a thermostable bacterial lipase in a novel yeast system.

A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.

Production and Characterization of Cross-Linked Aggregates of Geobacillus thermoleovorans CCR11 Thermoalkaliphilic Recombinant Lipase.

Immobilization of enzymes has many advantages for their application in biotechnological processes. In particular, the cross-linked enzyme aggregates (CLEAs) allow the production of solid biocatalysts with a high enzymatic loading and the advantage of obtaining derivatives with high stability at low cost. The purpose of this study was to produce cross-linked enzymatic aggregates (CLEAs) of LipMatCCR11, a 43 kDa recombinant solvent-tolerant thermoalkaliphilic lipase from Geobacillus thermoleovorans CCR11. LipMatCCR11-CLEAs were prepared using (NH4)2SO4 (40% w/v) as precipitant agent and glutaraldehyde (40 mM) as cross-linker, at pH 9, 20 °C. A U10(56) uniform design was used to optimize CLEA production, varying protein concentration, ammonium sulfate %, pH, glutaraldehyde concentration, temperature, and incubation time. The synthesized CLEAs were also analyzed using scanning electron microscopy (SEM) that showed individual particles of <1 µm grouped to form a superstructure. The cross-linked aggregates showed a maximum mass activity of 7750 U/g at 40 °C and pH 8 and retained more than 20% activity at 100 °C. Greater thermostability, resistance to alkaline conditions and the presence of organic solvents, and better durability during storage were observed for LipMatCCR11-CLEAs in comparison with the soluble enzyme. LipMatCCR11-CLEAs presented good reusability by conserving 40% of their initial activity after 9 cycles of reuse.

Chemical flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins.

A heterobimetallic Mn/Fe cofactor is present in the R2 subunit of class Ic ribonucleotide reductases (R2c) and in R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (µ-oxo/µ-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (µ-hydroxo) and a fatty acid ligand. Here, we identified a second coordination sphere residue that directs the divergent reactivity of the protein scaffold. We found that the residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group but not in R2c. Substitution of the glycine with leucine converted the resting-state R2lox cofactor to an R2c-like cofactor, a µ-oxo/µ-hydroxo-bridged MnIII/FeIII dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in WT R2lox, indicating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine-substituted variant no longer functions as a two-electron oxidant. Our results reveal that the residue preceding the N-terminal metal ligand directs the cofactor's reactivity toward one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.

Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the Ω Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover.

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]1+ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S-C5' bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5' of the 5'-deoxyadenosyl radical (5'-dAdo•). During catalysis, homolytic cleavage of the Fe-C5' bond liberates 5'-dAdo• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze-quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe-C5' bond of τ ≈ 5-6 s, likely shortened by enzymatic activation as is the case with the Co-C5' bond of B12. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5'-dAdo• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile• or Cys• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.

A systematic review of the genera Geobacillus and Parageobacillus: their evolution, current taxonomic status and major applications.

The genus Geobacillus, belonging to the phylum Firmicutes, is one of the most important genera and comprises thermophilic bacteria. The genus Geobacillus was erected with the taxonomic reclassification of various Bacillus species. Taxonomic studies of Geobacillus remain in progress. However, there is no comprehensive review of the characteristic features, taxonomic status and study of various applications of this interesting genus. The main aim of this review is to give a comprehensive account of the genus Geobacillus. At present the genus acomprises 25 taxa, 14 validly published (with correct name), nine validly published (with synonyms) and two not validly published species. We describe only validly published species of the genera Geobacillus and Parageobacillus. Vegetative cells of Geobacillus species are Gram-strain-positive or -variable, rod-shaped, motile, endospore-forming, aerobic or facultatively anaerobic, obligately thermophilic and chemo-organotrophic. Growth occurs in the pH range 6.08.5 and a temperature of 37-75 °C. The major cellular fatty acids are iso-C15:o, iso-C16:0 and iso-C17:o. The main menaquinone type is MK-7. The G-+C content of the DNA ranges between 48.2 and 58 mol%. The genus Geobacillus is widely distributed in nature, being mostly found in many extreme locations such as hot springs, hydrothermal vents, marine trenches, hay composts, etc. Geobacillus species have been widely exploited in various industrial and biotechnological applications, and thus are promising candidates for further studies in the future.

Molecular characterization of a highly efficient and thermostable phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis.

Phosphoribosyl anthranilate isomerase is involved in the isomerization of phosphoribosyl anthranilate to 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate. In the present study, trpFGt, a gene encoding phosphoribosyl anthranilate isomerase from Geobacillus thermopakistaniensis, was cloned and expressed in Escherichia coli. The gene product, TrpFGt, was produced in E. coli in soluble and active form. Molecular characterization revealed that recombinant TrpFGt was highly efficient and stable. The apparent Vmax and Km values were 480 µmol min-1 mg-1 and 1.15 µM, respectively. The half-life of the enzyme was 90 min at 60 °C. Apart from thermostability, TrpFGt was highly stable against protein denaturants such as urea. There was no significant change in activity even after treatment with 8 M urea. To the best of our knowledge, TrpFGt, is the most active and stable phosphoribosyl anthranilate isomerase characterized to date and this is the first characterization of TrpF from the genus Geobacillus.

Heterologous gene expression and characterization of recombinant aspartate aminotransferase from Geobacillus thermopakistaniensis.

Aspartate aminotransferase catalyzes the transfer of an amino group from l-aspartate to α-oxoglutarate. A gene encoding aspartate aminotransferase, ASTGt, from Geobacillus thermopakistaniensis was cloned and expressed in Escherichia coli. The purified recombinant ASTGt exhibited highest activity at 65 °C and pH 7.0. The activity was dependent on pyridoxal phosphate but not on any metal ions. Stoichiometry of purified ASTGt demonstrated that 0.1 pyridoxal phosphate was attached per subunit of the enzyme. Determination of molecular weight by gel filtration chromatography indicated that ASTGt existed in a dimeric form in solution. Thermostability experiments showed no significant change in activity even after 16 h incubation at 65 °C. ASTGt exhibited apparent Vmax and Km values of 120 µmol min-1 mg-1 and 1.5 mM, respectively, against l-aspartate. Substrate specificity experiments indicated the highest relative activity against aspartate (100%) followed by tyrosine (27%) and proline (16%). To the best of our knowledge, this is the first report on cloning and characterization of an AST from genus Geobacillus.

Refolding, characterization, and dye decolorization ability of a highly thermostable laccase from Geobacillus sp. JS12.

A putative laccase gene (lacG) from Geobacillus sp. JS12 was cloned and expressed as a fusion protein with six histidine residues in Escherichia coli BL21 (DE3) cells, and the protein was primarily found in inclusion bodies. The resulting insoluble proteins were solubilized with 6 M guanidine HCl and refolded using an on-column refolding procedure. Ni-chelation affinity chromatography found the laccase to be a 30 kDa monomeric protein. Spectrophotometry and electron paramagnetic resonance (EPR) analysis indicated LacG as a multi-copper oxidase, with the usual laccase copper sites, Type 1, 2, and 3 Cu(II). The optimum pH for enzymatic activity was 3.0, 6.0, and 6.5 with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), guaiacol and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The recombinant protein displayed high thermostability, with a heat inactivation half-life of approximately 2 h at 95 °C, and an optimum temperature of 80 °C with 2,6-DMP. Catalytic efficiency (kcat/Km) showed that guaiacol and 2,6-DMP were highly oxidized by the enzyme. The enzymatic reaction was significantly enhanced by Co2+ and Mn2+, while activity was strongly inhibited in the presence of Fe2+, Zn2+, and thiol compounds. LacG decolorized 43% of Congo red and 14% of Malachite green, and the addition of ABTS as a redox mediator dramatically increased the dye decolorization efficiency.

Mono-PEGylation of a Thermostable Arginine-Depleting Enzyme for the Treatment of Lung Cancer.

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.

Recombinant Bacillus caldovelox Arginase Mutant (BCA-M) Induces Apoptosis, Autophagy, Cell Cycle Arrest and Growth Inhibition in Human Cervical Cancer Cells.

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.

Ion-Pair Interaction and Hydrogen Bonds as Main Features of Protein Thermostability in Mutated T1 Recombinant Lipase Originating from Geobacillus zalihae.

A comparative structure analysis between space- and an Earth-grown T1 recombinant lipase from Geobacillus zalihae had shown changes in the formation of hydrogen bonds and ion-pair interactions. Using the space-grown T1 lipase validated structure having incorporated said interactions, the recombinant T1 lipase was re-engineered to determine the changes brought by these interactions to the structure and stability of lipase. To understand the effects of mutation on T1 recombinant lipase, five mutants were developed from the structure of space-grown T1 lipase and biochemically characterized. The results demonstrate an increase in melting temperature up to 77.4 °C and 76.0 °C in E226D and D43E, respectively. Moreover, the mutated lipases D43E and E226D had additional hydrogen bonds and ion-pair interactions in their structures due to the improvement of stability, as observed in a longer half-life and an increased melting temperature. The biophysical study revealed differences in ß-Sheet percentage between less stable (T118N) and other mutants. As a conclusion, the comparative analysis of the tertiary structure and specific residues associated with ion-pair interactions and hydrogen bonds could be significant in revealing the thermostability of an enzyme with industrial importance.

New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations.

This study presents a new microbial lipolytic enzyme GD-95RM designed via random mutagenesis using previously characterized GD-95 lipase as a template. The improvement in activity of GD-95 lipase was caused by E100K, F154V and V174I mutations. Compared with GD-95 lipase, the GD-95RM lipase had 1.3-fold increased specific activity (2000 U/mg), demonstrated resistance to higher temperatures (75-85 °C), had fourfold increased Vmax towards p-NP dodecanoate and showed 2.5-fold lower KM for p-NP butyrate. It retained > 50% of its lipolytic activity when hydrolyzing short, medium and long acyl chain substrates at 30 °C and 55 °C reaction temperatures after 20 days' incubation with 25% of ethanol. GD-95RM also displayed long-term tolerance (40 d) to 5% NaCl, trisodium citrate, sodium perborate, urea, 0.1% boric acid, citric acid and Triton X-100. Moreover, oil hydrolysis and transesterification results revealed the capability of GD-95RM lipase to produce fatty acids or fatty acid esters through eco-friendly hydrolysis and transesterification reactions using a broad range of vegetable and fish oils, animal fat and different alcohols as substrates. GD-95RM lipase was successfully applied in synthesis reactions for ethyl oleate, octyl oleate and isoamyl oleate without giving to use additional reaction compounds or special reaction conditions.

X-ray structure and characterization of a thermostable lipase from Geobacillus thermoleovorans.

Thermo-alkalophilic bacterium, Geobacillus thermoleovorans secrets many enzymes including a 43 kDa extracellular lipase. Significant thermostability, organic solvent stability and wide substrate preferences for hydrolysis drew our attention to solve its structure by crystallography. The structure was solved by molecular replacement method and refined up to 2.14 Šresolution. Structure of the lipase showed an alpha-beta fold with 19 α-helices and 10 ß-sheets. The active site remains covered by a lid. One calcium and one zinc atom was found in the crystal. The structure showed a major difference (rmsd 5.6 Å) from its closest homolog in the amino acid region 191 to 203. Thermal unfolding of the lipase showed that the lipase is highly stable with Tm of 76 °C. 13C NMR spectra of products upon triglyceride hydrolysate revealed that the lipase hydrolyses at both sn-1 and sn-2 positions with equal efficiency.

Assembly of a heterodinuclear Mn/Fe cofactor is coupled to tyrosine-valine ether cross-link formation in the R2-like ligand-binding oxidase.

R2-like ligand-binding oxidases (R2lox) assemble a heterodinuclear Mn/Fe cofactor which performs reductive dioxygen (O2) activation, catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold, and binds a fatty acid in a putative substrate channel. We have previously shown that the N-terminal metal binding site 1 is unspecific for manganese or iron in the absence of O2, but prefers manganese in the presence of O2, whereas the C-terminal site 2 is specific for iron. Here, we analyze the effects of amino acid exchanges in the cofactor environment on cofactor assembly and metalation specificity using X-ray crystallography, X-ray absorption spectroscopy, and metal quantification. We find that exchange of either the cross-linking tyrosine or the valine, regardless of whether the mutation still allows cross-link formation or not, results in unspecific manganese or iron binding at site 1 both in the absence or presence of O2, while site 2 still prefers iron as in the wild-type. In contrast, a mutation that blocks binding of the fatty acid does not affect the metal specificity of either site under anoxic or aerobic conditions, and cross-link formation is still observed. All variants assemble a dinuclear trivalent metal cofactor in the aerobic resting state, independently of cross-link formation. These findings imply that the cross-link residues are required to achieve the preference for manganese in site 1 in the presence of O2. The metalation specificity, therefore, appears to be established during the redox reactions leading to cross-link formation.