A missense variant in SHARPIN mediates Alzheimer's disease-specific brain damages.

Established genetic risk factors for Alzheimer's disease (AD) account for only a portion of AD heritability. The aim of this study was to identify novel associations between genetic variants and AD-specific brain atrophy. We conducted genome-wide association studies for brain magnetic resonance imaging measures of hippocampal volume and entorhinal cortical thickness in 2643 Koreans meeting the clinical criteria for AD (n = 209), mild cognitive impairment (n = 1449) or normal cognition (n = 985). A missense variant, rs77359862 (R274W), in the SHANK-associated RH Domain Interactor (SHARPIN) gene was associated with entorhinal cortical thickness (p = 5.0 × 10-9) and hippocampal volume (p = 5.1 × 10-12). It revealed an increased risk of developing AD in the mediation analyses. This variant was also associated with amyloid-ß accumulation (p = 0.03) and measures of memory (p = 1.0 × 10-4) and executive function (p = 0.04). We also found significant association of other SHARPIN variants with hippocampal volume in the Alzheimer's Disease Neuroimaging Initiative (rs3417062, p = 4.1 × 10-6) and AddNeuroMed (rs138412600, p = 5.9 × 10-5) cohorts. Further, molecular dynamics simulations and co-immunoprecipitation indicated that the variant significantly reduced the binding of linear ubiquitination assembly complex proteins, SHPARIN and HOIL-1 Interacting Protein (HOIP), altering the downstream NF-κB signaling pathway. These findings suggest that SHARPIN plays an important role in the pathogenesis of AD.

Functional and structural characterization of Deinococcus radiodurans R1 MazEF toxin-antitoxin system, Dr0416-Dr0417.

In prokaryotes, toxin-antitoxin (TA) systems are commonly found. They likely reflect the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by slowing their growth rate. Genomic analysis of the extremophile Deinococcus radiodurans R1 revealed the presence of eight type II TA systems, including the genes dr0417, dr0660, dr1530, dr0690, and dr1807. Expression of these toxin genes led to inhibition of Escherichia coli growth, whereas their antidote antitoxins were able to recover the growth defect. Remarkably, Dr0417 (DrMazF) showed endoribonuclease activity toward rRNAs as well as mRNAs, as determined by in vivo and in vitro RNA cleavage assays, and this activity was inhibited by Dr0416 (DrMazE). It was also found that the expression of dr0416-0417 module is directly regulated by the DrMazE-MazF complex. Furthermore, this TA module was induced under stress conditions and plays an important role in survival. To understand the regulatory mechanism at the molecular level, we determined the first high-resolution structures of DrMazF alone and of the DrMazE-MazF complex. In contrast with the hetero-hexameric state of typical MazE-MazF complexes found in other species, DrMazE-MazF crystal structure consists of a hetero-trimer, with the DNA-binding domain of DrMazE undergoing self-cleavage at the flexible linker loop. Our structure revealed that the unique residue R54 provides an additional positive charge to the substrate-binding pocket of DrMazF, its mutation significantly affects the endonuclease activity. Thus, our work reports the unique structural and biochemical features of the DrMazE-MazF system.

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.

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.

Non-catalytic-Region Mutations Conferring Transition of Class A ß-Lactamases Into ESBLs.

Despite class A ESBLs carrying substitutions outside catalytic regions, such as Cys69Tyr or Asn136Asp, have emerged as new clinical threats, the molecular mechanisms underlying their acquired antibiotics-hydrolytic activity remains unclear. We discovered that this non-catalytic-region (NCR) mutations induce significant dislocation of ß3-ß4 strands, conformational changes in critical residues associated with ligand binding to the lid domain, dynamic fluctuation of Ω-loop and ß3-ß4 elements. Such structural changes increase catalytic regions' flexibility, enlarge active site, and thereby accommodate third-generation cephalosporin antibiotics, ceftazidime (CAZ). Notably, the electrostatic property around the oxyanion hole of Cys69Tyr ESBL is significantly changed, resulting in possible additional stabilization of the acyl-enzyme intermediate. Interestingly, the NCR mutations are as effective for antibiotic resistance by altering the structure and dynamics in regions mediating substrate recognition and binding as single amino-acid substitutions in the catalytic region of the canonical ESBLs. We believe that our findings are crucial in developing successful therapeutic strategies against diverse class A ESBLs, including the new NCR-ESBLs.

Crystal Structure of Cytochrome cL from the Aquatic Methylotrophic Bacterium Methylophaga aminisulfidivorans MPT.

Cytochrome cL (CytcL) is an essential protein in the process of methanol oxidation in methylotrophs. It receives an electron from the pyrroloquinoline quinone (PQQ) cofactor of methanol dehydrogenase (MDH) to produce formaldehyde. The direct electron transfer mechanism between CytcL and MDH remains unknown due to the lack of structural information. To help gain a better understanding of the mechanism, we determined the first crystal structure of heme c containing CytcL from the aquatic methylotrophic bacterium Methylophaga aminisulfidivorans MPT at 2.13 Å resolution. The crystal structure of Ma-CytcL revealed its unique features compared to those of the terrestrial homologues. Apart from Fe in heme, three additional metal ion binding sites for Na+ , Ca+ , and Fe2+ were found, wherein the ions mostly formed coordination bonds with the amino acid residues on the loop (G93-Y111) that interacts with heme. Therefore, these ions seemed to enhance the stability of heme insertion by increasing the loop's steadiness. The basic N-terminal end, together with helix α4 and loop (G126 to Y136), contributed positive charge to the region. In contrast, the acidic C-terminal end provided a negatively charged surface, yielding several electrostatic contact points with partner proteins for electron transfer. These exceptional features of Ma-CytcL, along with the structural information of MDH, led us to hypothesize the need for an adapter protein bridging MDH to CytcL within appropriate proximity for electron transfer. With this knowledge in mind, the methanol oxidation complex reconstitution in vitro could be utilized to produce metabolic intermediates at the industry level.

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 ß-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-ß-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.

Correction to: Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1.

Following publication of the original article [1], the authors have re-evaluated the authorship for this article. The updated author group is.

SPIN90, an adaptor protein, alters the proximity between Rab5 and Gapex5 and facilitates Rab5 activation during EGF endocytosis.

During ligand-mediated receptor endocytosis, the small GTPase Rab5 functions in vesicle fusion and trafficking. Rab5 activation is known to require interactions with its guanine nucleotide-exchange factors (GEFs); however, the mechanism regulating Rab5 interactions with GEFs remains unclear. Here, we show that the SH3-adapter protein SPIN90 participates in the activation of Rab5 through the recruitment of both Rab5 and its GEF, Gapex5, to endosomal membranes during epidermal growth factor (EGF)-mediated endocytosis. SPIN90 strongly interacts with the inactive Rab5/GDI2 complex through its C-terminus. In response to EGF signaling, extracellular signal-regulated kinase (ERK)-mediated phosphorylation of SPIN90 at Thr-242 enables SPIN90 to bind Gapex5 through its N-terminal SH3 domain. Gapex5 is a determinant of Rab5 membrane targeting, while SPIN90 mediates the interaction between Gapex5 and Rab5 in a phosphorylation-dependent manner. Collectively, our findings suggest that SPIN90, as an adaptor protein, simultaneously binds inactive Rab5 and Gapex5, thereby altering their spatial proximity and facilitating Rab5 activation.

The crystal structure of methanol dehydrogenase, a quinoprotein from the marine methylotrophic bacterium Methylophaga aminisulfidivorans MPT.

The first crystal structure of a pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) from a marine methylotrophic bacterium, Methylophaga aminisulfidivorans MPT (MDH Mas ), was determined at 1.7 Å resolution. The active form of MDH Mas (or MDHI Mas ) is a heterotetrameric α2ß2, where each ß-subunit assembles on one side of each of the α-subunits, in a symmetrical fashion, so that two ß-subunits surround the two PQQ-binding pockets on the α-subunits. The active site consists of a PQQ molecule surrounded by a ß-propeller fold for each α-subunit. Interestingly, the PQQ molecules are coordinated by a Mg2+ ion, instead of the Ca2+ ion that is commonly found in the terrestrial MDHI, indicating the efficiency of osmotic balance regulation in the high salt environment. The overall interaction of the ß-subunits with the α-subunits appears tighter than that of terrestrial homologues, suggesting the efficient maintenance of MDHI Mas integrity in the sea water environment to provide a firm basis for complex formation with MxaJ Mas or Cyt cL. With the help of the features mentioned above, our research may enable the elucidation of the full molecular mechanism of methanol oxidation by taking advantage of marine bacterium-originated proteins in the methanol oxidizing system (mox), including MxaJ, as the attainment of these proteins from terrestrial bacteria for structural studies has not been successful.

Erratum to: Potentiation of TRAIL killing activity by multimerization through isoleucine zipper hexamerization motif.

The BMB Reports would like to correct in the ACKNOWLEDGEMENTS of BMB Rep. 49(5), 282-287 titled "Potentiation of TRAIL killing activity by multimerization through isoleucine zipper hexamerization motif."

TM0416, a Hyperthermophilic Promiscuous Nonphosphorylated Sugar Isomerase, Catalyzes Various C5 and C6 Epimerization Reactions.

There is currently little information on nonphosphorylated sugar epimerases, which are of potential interest for producing rare sugars. We found a gene (the TM0416 gene) encoding a putative d-tagatose-3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima We overexpressed the TM0416 gene in Escherichia coli and purified the resulting recombinant protein for detailed characterization. Amino acid sequence alignment and a structural similarity search revealed that TM0416 is a putative nonphosphorylated sugar epimerase. The recombinant enzyme exhibited maximal C-3 epimerization of l-ribulose to l-xylulose at ∼80°C and pH 7 in the presence of 1 mM Mn2+ In addition, this enzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose, with a conversion yield of 20% after 6 h at 80°C. Remarkably, the enzyme catalyzed the isomerization of d-erythrose or d-threose to d-erythrulose significantly, with conversion yields of 71% and 54.5%, respectively, after 6 h at 80°C at pH 7. To further investigate the substrate specificity of TM0416, we determined its crystal structures in complex with divalent metal ions and l-erythrulose at resolutions of 1.5 and 1.6 Å. Detailed inspection of the structural features and biochemical data clearly demonstrated that this metalloenzyme, with a freely accessible substrate-binding site and neighboring hydrophobic residues, exhibits different and promiscuous substrate preferences, compared with its mesophilic counterparts. Therefore, this study suggests that TM0416 can be functionally classified as a novel type of l-ribulose 3-epimerase (R3E) with d-erythrose isomerase activity.IMPORTANCE Rare sugars, which occur naturally in small amounts, have attracted considerable attention in the food and drug industries. However, there is little information on nonphosphorylated sugar epimerases, which might potentially be applied for the production of rare sugars. This study describes the characterization and functional annotation of a putative nonphosphorylated sugar 3-epimerase from a hyperthermophilic bacterium. Furthermore, we determined its crystal structures in complex with divalent metal ions and l-erythrulose, highlighting its metal-dependent, bifunctional, sugar-isomerizing activity. This hyperthermophilic R3E exhibited d-erythrose/d-threose isomerase activity, with structural features near the substrate-binding site distinct from those of its mesophilic counterparts. Moreover, this metalloenzyme showed unusually high activity for the epimerization of d-tagatose to d-sorbose at 70°C. Therefore, TM0416 can be functionally classified as a novel type of promiscuous R3E with a potential for the production of rare sugars for the food and pharmaceutical industries.

Dimerization in LBD16 and LBD18 Transcription Factors Is Critical for Lateral Root Formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (hereafter referred to as LBD) are plant-specific transcription factors that play important roles in a plethora of plant growth and development. The leucine (Leu) zipper-like coiled-coil motif in the lateral organ boundaries domain of the class I LBD proteins has been proposed to mediate protein dimerization, but it has not been experimentally assessed yet. LBD16 and LBD18 have been well characterized to play important roles in lateral root development in Arabidopsis (Arabidopsis thaliana). Here, we investigated the role of the coiled-coil motif in the dimerization of LBD16 and LBD18 and in transcriptional regulation and biological function. We built the molecular models of the coiled coil of LBD16 and LBD18, providing the probable Leu zipper models of the helix dimer. Using a variety of molecular techniques, such as bimolecular fluorescence complementation, luciferase complementation imaging, GST pull down, and coimmunoprecipitation assays, we showed that the conserved Leu or valine residues in the coiled-coil motif are critical for the dimerization of LBD16 or LBD18. Using transgenic Arabidopsis plants that overexpress HA:LBD16 or HA:LBD16Q in lbd16 or HA:LBD18 or HA:LBD18Q in lbd18, we demonstrated that the homodimerization of LBD18 mediated by the coiled-coil motif is crucial for transcriptional regulation via promoter binding and for lateral root formation. In addition, we found that the carboxyl-terminal region beyond the coiled-coil motif in LBD18 acts as an additional dimerization domain. These results provide a molecular basis for homodimerization and heterodimerization among the 42 Arabidopsis LBD family members for displaying their biological functions.

Structural implications of Ca2+-dependent actin-bundling function of human EFhd2/Swiprosin-1.

EFhd2/Swiprosin-1 is a cytoskeletal Ca2+-binding protein implicated in Ca2+-dependent cell spreading and migration in epithelial cells. EFhd2 domain architecture includes an N-terminal disordered region, a PxxP motif, two EF-hands, a ligand mimic helix and a C-terminal coiled-coil domain. We reported previously that EFhd2 displays F-actin bundling activity in the presence of Ca2+ and this activity depends on the coiled-coil domain and direct interaction of the EFhd2 core region. However, the molecular mechanism for the regulation of F-actin binding and bundling by EFhd2 is unknown. Here, the Ca2+-bound crystal structure of the EFhd2 core region is presented and structures of mutants defective for Ca2+-binding are also described. These structures and biochemical analyses reveal that the F-actin bundling activity of EFhd2 depends on the structural rigidity of F-actin binding sites conferred by binding of the EF-hands to Ca2+. In the absence of Ca2+, the EFhd2 core region exhibits local conformational flexibility around the EF-hand domain and C-terminal linker, which retains F-actin binding activity but loses the ability to bundle F-actin. In addition, we establish that dimerisation of EFhd2 via the C-terminal coiled-coil domain, which is necessary for F-actin bundling, occurs through the parallel coiled-coil interaction.

High adaptability of the omega loop underlies the substrate-spectrum-extension evolution of a class A ß-lactamase, PenL.

The omega loop in ß-lactamases plays a pivotal role in substrate recognition and catalysis, and some mutations in this loop affect the adaptability of the enzymes to new antibiotics. Various mutations, including substitutions, deletions, and intragenic duplications resulting in tandem repeats (TRs), have been associated with ß-lactamase substrate spectrum extension. TRs are unique among the mutations as they cause severe structural perturbations in the enzymes. We explored the process by which TRs are accommodated in order to test the adaptability of the omega loop. Structures of the mutant enzymes showed that the extra amino acid residues in the omega loop were freed outward from the enzyme, thereby maintaining the overall enzyme integrity. This structural adjustment was accompanied by disruptions of the internal α-helix and hydrogen bonds that originally maintained the conformation of the omega loop and the active site. Consequently, the mutant enzymes had a relaxed binding cavity, allowing for access of new substrates, which regrouped upon substrate binding in an induced-fit manner for subsequent hydrolytic reactions. Together, the data demonstrate that the design of the binding cavity, including the omega loop with its enormous adaptive capacity, is the foundation of the continuous evolution of ß-lactamases against new drugs.

Structural insight for substrate tolerance to 2-deoxyribose-5-phosphate aldolase from the pathogen Streptococcus suis.

2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/ß)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and ß8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility.

Structure of the thermophilic l-Arabinose isomerase from Geobacillus kaustophilus reveals metal-mediated intersubunit interactions for activity and thermostability.

Thermophilic l-arabinose isomerase (AI), which catalyzes the interconversion of l-arabinose and l-ribulose, can be used to produce d-tagatose, a sugar substitute, from d-galactose. Unlike mesophilic AIs, thermophilic AIs are highly dependent on divalent metal ions for their catalytic activity and thermostability at elevated temperatures. However, the molecular basis underlying the substrate preferences and metal requirements of multimeric AIs remains unclear. Here we report the first crystal structure of the apo and holo forms of thermophilic Geobacillus kaustophilus AI (GKAI) in hexamer form. The structures, including those of GKAI in complex with l-arabitol, and biochemical analyses revealed not only how the substrate-binding site of GKAI is formed through displacement of residues at the intersubunit interface when it is bound to Mn(2+), but also revealed the water-mediated H-bonding networks that contribute to the structural integrity of GKAI during catalysis. These observations suggest metal-mediated isomerization reactions brought about by intersubunit interactions at elevated temperatures are responsible for the distinct active site features that promote the substrate specificity and thermostability of thermophilic AIs.

Potentiation of TRAIL killing activity by multimerization through isoleucine zipper hexamerization motif.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a homo-trimeric cytotoxic ligand. Several studies have demonstrated that incorporation of artificial trimerization motifs into the TRAIL protein leads to the enhancement of biological activity. Here, we show that linkage of the isoleucine zipper hexamerization motif to the N-terminus of TRAIL, referred as ILz(6):TRAIL, leads to multimerization of its trimeric form, which has higher cytotoxic activity compared to its native state. Size exclusion chromatography of ILz(6):TRAIL revealed possible existence of various forms such as trimeric, hexameric, and multimeric (possibly containing one-, two-, and multi-units of trimeric TRAIL, respectively). Increased number of multimerized ILz(6):TRAIL units corresponded with enhanced cytotoxic activity. Further, a high degree of ILz(6):TRAIL multimerization triggered rapid signaling events such as activation of caspases, tBid generation, and chromatin condensation. Taken together, these results indicate that multimerization of TRAIL significantly enhances its cytotoxic activity. [BMB Reports 2016; 49(5): 282-287].

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 Co(2+). 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.

The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.

UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism.