Fervidobacterium Islandicum AW-1 Research Articles

Structure of oxidized pyrrolidone carboxypeptidase from Fervidobacterium islandicum AW-1 reveals unique structural features for thermostability and keratinolysis

Pyrrolidone carboxypeptidases (Pcps) (E.C. 3.4.19.3) can cleave the peptide bond adjacent to pyro-glutamic acid (pGlu), an N-terminal modification observed in some proteins that provides protection against common proteases. Pcp derived from extremely thermophilic Fervidobacterium islandicum AW-1 (FiPcp), that belongs to the cysteine protease family, is involved in keratin utilization under stress conditions. Although an irreversible oxidative modification of active cysteine to its sulfonic acid derivative (Cys-SO3H) renders the enzyme inactive, the molecular details for the sulfonic acid modification in inactive Pcp remain unclear. Here, we determined the crystal structure of FiPcp at 1.85 Å, revealing the oxidized form of cysteine sulfonic acid (C156–SO3H) in the catalytic triad (His-Cys-Glu), which participates in the hydrolysis of pGlu residue containing peptide bond. The three oxygen atoms of cysteine sulfonic acid were stabilized by hydrogen bonds with H180, carbonyl backbone of Q83, and water molecules, resulting in inactivation of FiPcp. Furthermore, FiPcp demonstrated a unique 139KKKK142 motif involved in inter-subunit electrostatic interactions whose mutation significantly affects the thermostability of tetrameric FiPcp. Thus, our high-resolution structure of the first inactive FiPcp with irreversible oxidative modification of active cysteine provides not only the molecular basis of the redox-dependent catalysis of Pcp, but also the structural features of its thermostability.

Functional Characterization of Primordial Protein Repair Enzyme M38 Metallo-Peptidase From Fervidobacterium islandicum AW-1.

The NA23_RS08100 gene of Fervidobacterium islandicum AW-1 encodes a keratin-degrading β-aspartyl peptidase (FiBAP) that is highly expressed under starvation conditions. Herein, we expressed the gene in Escherichia coli, purified the recombinant enzyme to homogeneity, and investigated its function. The 318 kDa recombinant FiBAP enzyme exhibited maximal activity at 80°C and pH 7.0 in the presence of Zn2+. Size-exclusion chromatography revealed that the native enzyme is an octamer comprising a tetramer of dimers; this was further supported by determination of its crystal structure at 2.6 Å resolution. Consistently, the structure of FiBAP revealed three additional salt bridges in each dimer, involving 12 ionic interactions that might contribute to its high thermostability. In addition, the co-crystal structure containing the substrate analog N-carbobenzoxy-β-Asp-Leu at 2.7 Å resolution revealed binuclear Zn2+-mediated substrate binding, suggesting that FiBAP is a hyperthermophilic type-I IadA, in accordance with sequence-based phylogenetic analysis. Indeed, complementation of a Leu auxotrophic E. coli mutant strain (ΔiadA and ΔleuB) with FiBAP enabled the mutant strain to grow on isoAsp-Leu peptides. Remarkably, LC-MS/MS analysis of soluble keratin hydrolysates revealed that FiBAP not only cleaves the C-terminus of isoAsp residues but also has a relatively broad substrate specificity toward α-peptide bonds. Moreover, heat shock-induced protein aggregates retarded bacterial growth, but expression of BAP alleviated the growth defect by degrading damaged proteins. Taken together, these results suggest that the viability of hyperthermophiles under stressful conditions may rely on the activity of BAP within cellular protein repair systems.

The sulfur formation system mediating extracellular cysteine-cystine recycling in Fervidobacterium islandicum AW-1 is associated with keratin degradation.

Most extremophilic anaerobes possess a sulfur formation (Suf) system for Fe-S cluster biogenesis. In addition to its essential role in redox chemistry and stress responses of Fe-S cluster proteins, the Suf system may play an important role in keratin degradation by Fervidobacterium islandicum AW-1. Comparative genomics of the order Thermotogales revealed that the feather-degrading F. islandicum AW-1 has a complete Suf-like machinery (SufCBDSU) that is highly expressed in cells grown on native feathers in the absence of elemental sulfur (S0 ). On the other hand, F. islandicum AW-1 exhibited a significant retardation in the Suf system-mediated keratin degradation in the presence of S0 . Detailed differential expression analysis of sulfur assimilation machineries unveiled the mechanism by which an efficient sulfur delivery from persulfurated SufS to SufU is achieved during keratinolysis under sulfur starvation. Indeed, addition of SufS-SufU to cell extracts containing keratinolytic proteases accelerated keratin decomposition in vitro under reducing conditions. Remarkably, mass spectrometric analysis of extracellular and intracellular levels of amino acids suggested that redox homeostasis within cells coupled to extracellular cysteine and cystine recycling might be a prerequisite for keratinolysis. Taken together, these results suggest that the Suf-like machinery including the SufS-SufU complex may contribute to sulfur availability for an extracellular reducing environment as well as intracellular redox homeostasis through cysteine released from keratin hydrolysate under starvation conditions.

Crystallization and preliminary X-ray diffraction analysis of Thioredoxin from the feather-degrading thermophile Fervidobacterium islandicum AW-1

Crystallization and preliminary X-ray diffraction analysis of Thioredoxin from the feather-degrading thermophile Fervidobacterium islandicum AW-1

Fluorescence-based Quantification of Bioactive Keratin Peptides from Feathers for Optimizing Large-scale Anaerobic Fermentation and Purification

The extremely thermophilic eubacterium Fervidobacterium islandicum AW-1 produces low molecular weight (LMW; < 1 kDa) keratin peptides (KPs) from poultry feathers at 70°C. However, detection and quantification of feather hydrolysate-derived peptides is needed for optimizing fermentation and down-stream processes. Herein, we developed a large-scale fermentation and purification of skin anti-aging LMW KPs from recalcitrant feathers using fluorescence-based quantification of N-terminal prolinecontaining KPs derivatized with 3,4-dihydroxybenzoic acid to yield fluorescent adducts. Fluorescent products were correlated with bioactive KP concentrations in keratin fractions and cosmetic formulations. Subsequent anaerobic fermentative keratinolysis and large-scale purification achieved 4.4 g/L LMW KPs from 8 g/L native feathers in a 5 L batch bioreactor, generating 0.8 g/L purified MMP-1 suppressive KPs (yield = 1.2%). This demonstrated the feasibility of industrial-scale anaerobic feather digestion and purification of LMW KPs to produce skin anti-aging peptides from keratin hydrolysates in a more environmentally sustainable manner.

Low-molecular weight keratins with anti-skin aging activity produced by anaerobic digestion of poultry feathers with Fervidobacterium islandicum AW-1

Bioactive peptides contribute to various cellular processes including improved skin physiology. Hence, bioactive keratins have attracted considerable attention as active cosmetic ingredients for skin health. Here, we obtained low molecular weight (LMW) keratins from native chicken feathers by anaerobic digestion with an extremely thermophilic bacterium Fervidobacterium islandicum AW-1, followed by stepwise fractionation through ultrafiltration. To assess the effects of the feather keratins on skin health, we performed in vitro and ex vivo assays to investigate their inhibitory effects on matrix metalloproteinases (MMPs). As results, LMW feather keratins marginally inhibited collagenase, elastase, and radical scavenging activities. On the other hand, LMW feather keratins significantly suppressed the expression of ultraviolet B (UVB)-induced MMP-1 and MMP-13 in human dermal fibroblasts. Furthermore, phospho-kinase antibody array revealed that LMW feather keratins suppressed UVB-induced phosphorylation of Akts, c-Jun N-terminal kinases 1, p38 beta, and RSK2, but not ERKs in human dermal fibroblast. Overall, these results suggest that LMW feather keratins are potential candidates as cosmeceutical peptides for anti-skin aging.

Development of a keratinase activity assay using recombinant chicken feather keratin substrates.

Poultry feathers consist mainly of the protein keratin, which is rich in β-pleated sheets and consequently resistant to proteolysis. Although many keratinases have been identified, the reasons for their substrate specificity towards β-keratin remain unclear due to difficulties in preparing a soluble feather keratin substrate for use in activity assays. In the present study, we overexpressed Gallus gallus chromosomes 2 and 27 β-keratin-encoding genes in Escherichia coli, purified denatured recombinant proteins by Ni2+ affinity chromatography, and refolded by stepwise dialysis to yield soluble keratins. To assess the keratinolytic activity, we compared the proteolytic activity of crude extracts from the feather- degrading bacterium Fervidobacterium islandicum AW-1 with proteinase K, trypsin, and papain using purified recombinant keratin and casein as substrates. All tested proteases showed strong proteolytic activities for casein, whereas only F. islandicum AW-1 crude extracts and proteinase K exhibited pronounced keratinolytic activity for the recombinant keratin. Moreover, LC-MS/MS analysis of keratin hydrolysates allowed us to predict the P1 sites of keratinolytic enzymes in the F. islandicum AW-1 extracts, thereby qualifying and quantifying the extent of keratinolysis. The soluble keratin-based assay has clear therapeutic and industrial potential for the development of a high-throughput screening system for proteases hydrolyzing disease-related protein aggregates, as well as mechanically resilient keratin-based polymers.

Enzymatic Characteristics of a Highly Thermostable β-(1-4)-Glucanase from Fervidobacterium islandicum AW-1 (KCTC 4680).

A highly thermostable β-(1-4)-glucanase (NA23_08975) gene (fig) from Fervidobacterium islandicum AW-1, a native-feather degrading thermophilic eubacterium, was cloned and expressed in Escherichia coli. The recombinant FiG (rFiG) protein showed strong activity toward β-D-glucan from barley (367.0 IU/mg), galactomannan (174.0 IU/mg), and 4-nitrophenyl-cellobioside (66.1 IU/mg), but relatively weak activity was observed with hydroxyethyl cellulose (5.3 IU/mg), carboxymethyl cellulose (2.4 IU/mg), and xylan from oat spelt (1.4 IU/mg). rFiG exhibited optimal activity at 90°C and pH 5.0. In addition, this enzyme was extremely thermostable, showing a half-life of 113 h at 85°C. These results indicate that rFiG could be used for hydrolysis of cellulosic and hemicellulosic biomass substrates for biofuel production.

Biochemical and structural characterization of a keratin-degrading M32 carboxypeptidase from Fervidobacterium islandicum AW-1

Comparative genomics of the keratin-degrading extremophilic eubacterium Fervidobacterium islandicum AW-1 and the closely related Fervidobacterium nodosum with no keratinolytic activity suggested that the FIAW1_1600 gene encoding a carboxypeptidase (CP) plays an important role in keratin degradation. The presumptive 489 amino acid sequence of the gene showed a conserved HEXXH motif with low levels of sequence identity (<38%) to reported thermostable M32 CPs. To identify its functional role, the FIAW1_1600 gene was overexpressed in Escherichia coli, and the recombinant enzyme was purified and characterized in detail. F. islandicum AW-1 CP (FisCP) formed a homodimer with a molecular mass of 107 kDa, and its apoenzyme exhibited maximal activity at 80 °C and pH 7.0 in the presence of Co2+. This metalloenzyme mainly cleaved the C-termini of peptides with a basic amino acid sequence. The crystal structure of FisCP at 2.2 Å resolution showed high levels of structural similarities (root-mean-square deviations of <1.7 Å) to those of other M32 CP homologs. Remarkably, the enzyme significantly enhanced the degradation of native chicken feathers. This study suggests that FisCP, a keratinolytic member of the thermostable M32 CP family, plays an important role in keratin degradation for cellular metabolism in F. islandicum AW-1.

Native-feather degradation by Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing thermophilic anaerobe.

A native-feather-degrading thermophilic anaerobe was isolated from a geothermal hot stream in Indonesia. Isolate AW-1, identified as a member of the species Fervidobacterium islandicum, was shown to degrade native feathers (0.8%, w/v) completely at 70 degrees C and pH 7 with a maximum specific growth rate (0.14 h(-1)) in Thermotoga- Fervidobacterium(TF) medium. After 24 h of culture, feather degradation led to an increase in free amino acids such as histidine, cysteine and lysine. Moreover, nutritionally essential amino acids such as tryptophan and methionine, which are rare in feather keratin, were also produced as microbial metabolites. A homomultimeric membrane-bound keratinolytic protease (>200 kDa; 97 kDa subunits) was purified from a cell extract of F. islandicum AW-1. The enzyme exhibited activity toward casein and soluble keratin optimally at 100 degrees C and pH 9, and had a half-life of 90 min at 100 degrees C. The enzyme showed higher specific activity for the keratinous substrates than other proteases and catalyzed the cleavage of peptide bonds more rapidly following the reduction of disulfide bridges in feather keratin by 10 mM dithiothreitol. Therefore, the enzyme from F. islandicum AW-1 is a novel, thermostable keratinolytic serine protease.