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The crystal structure of CYP105D18 and its unique structural features for papaverine N-oxidation are presented., The bacterial CYP105 family is involved in secondary metabolite biosynthetic pathways and plays essential roles in the biotransformation of xenobiotics. This study investigates the newly identified H2O2-mediated CYP105D18 from Streptomyces laurentii as the first bacterial CYP for N-oxidation. The catalytic efficiency of CYP105D18 for papaverine N-oxidation was 1.43 s−1 µM −1. The heme oxidation rate (k) was low (

The expression of aminoglycoside-modifying enzymes represents a survival strategy of antibiotic-resistant bacteria. Aminoglycoside 2′-N-acetyltransferase [AAC(2′)] neutralizes aminoglycoside drugs by acetylation of their 2′ amino groups in an acetyl coenzyme A (CoA)-dependent manner. To understand the structural features and molecular mechanism underlying AAC(2′) activity, we overexpressed, purified, and crystallized AAC(2′) from Mycolicibacterium smegmatis [AAC(2′)-Id] and determined the crystal structures of its apo-form and ternary complexes with CoA and four different aminoglycosides (gentamicin, sisomicin, neomycin, and paromomycin). These AAC(2′)-Id structures unraveled the binding modes of different aminoglycosides, explaining the broad substrate specificity of the enzyme. Comparative structural analysis showed that the α4-helix and β8–β9 loop region undergo major conformational changes upon CoA and substrate binding. Additionally, structural comparison between the present paromomycin-bound AAC(2′)-Id structure and the previously reported paromomycin-bound AAC(6′)-Ib and 30S ribosome structures revealed the structural features of paromomycin that are responsible for its antibiotic activity and AAC binding. Taken together, these results provide useful information for designing AAC(2′) inhibitors and for the chemical modification of aminoglycosides.

Crystal structures of enoyl-coenzyme A (CoA) isomerase from Bosea sp. PAMC 26642 (BoECI) and enoyl-CoA hydratase from Hymenobacter sp. PAMC 26628 (HyECH) were determined at 2.35 and 2.70 Å resolution, respectively. BoECI and HyECH are members of the crotonase superfamily and are enzymes known to be involved in fatty acid degradation. Structurally, these enzymes are highly similar except for the orientation of their C-terminal helix domain. Analytical ultracentrifugation was performed to determine the oligomerization states of BoECI and HyECH revealing they exist as trimers in solution. However, their putative ligand-binding sites and active site residue compositions are dissimilar. Comparative sequence and structural analysis revealed that the active site of BoECI had one glutamate residue (Glu135), this site is occupied by an aspartate in some ECIs, and the active sites of HyECH had two highly conserved glutamate residues (Glu118 and Glu138). Moreover, HyECH possesses a salt bridge interaction between Glu98 and Arg152 near the active site. This interaction may allow the catalytic Glu118 residue to have a specific conformation for the ECH enzyme reaction. This salt bridge interaction is highly conserved in known bacterial ECH structures and ECI enzymes do not have this type of interaction. Collectively, our comparative sequential and structural studies have provided useful information to distinguish and classify two similar bacterial crotonase superfamily enzymes.

Cells regulate their intracellular mRNA levels by using specific ribonucleases. Oligoribonuclease (ORN) is a 3′–5′ exoribonuclease for small RNA molecules, important in RNA degradation and re-utilisation. However, there is no structural information on the ligand-binding form of ORNs. In this study, the crystal structures of oligoribonuclease from Colwellia psychrerythraea strain 34H (CpsORN) were determined in four different forms: unliganded-structure, thymidine 5′-monophosphate p-nitrophenyl ester (pNP-TMP)-bound, two separated uridine-bound, and two linked uridine (U-U)-bound forms. The crystal structures show that CpsORN is a tight dimer, with two separated active sites and one divalent metal cation ion in each active site. These structures represent several snapshots of the enzymatic reaction process, which allowed us to suggest a possible one-metal-dependent reaction mechanism for CpsORN. Moreover, the biochemical data support our suggested mechanism and identified the key residues responsible for enzymatic catalysis of CpsORN.

In spore forming microbes, germination protease (GPR) plays a key role in the initiation of the germination process. A critical step during germination is the degradation of small acid-soluble proteins (SASPs), which protect spore DNA from external stresses (UV, heat, low temperature, etc.). Inactive zymogen GPR can be activated by autoprocessing of the N-terminal pro-sequence domain. Activated GPR initiates the degradation of SASPs; however, the detailed mechanisms underlying the activation, catalysis, regulation, and substrate recognition of GPR remain elusive. In this study, we determined the crystal structure of GPR from Paenisporosarcina sp. TG-20 (PaGPR) in its inactive form at a resolution of 2.5 A. Structural analysis showed that the active site of PaGPR is sterically occluded by an inhibitory loop region (residues 202-216). The N-terminal region interacts directly with the self-inhibitory loop region, suggesting that the removal of the N-terminal pro-sequence induces conformational changes, which lead to the release of the self-inhibitory loop region from the active site. In addition, comparative sequence and structural analyses revealed that PaGPR contains two highly conserved Asp residues (D123 and D182) in the active site, similar to the putative aspartic acid protease GPR from Bacillus megaterium. The catalytic domain structure of PaGPR also shares similarities with the sequentially non-homologous proteins HycI and HybD. HycI and HybD are metal-loproteases that also contain two Asp (or Glu) residues in their active site, playing a role in metal binding. In summary, our results provide useful insights into the activation process of PaGPR and its active conformation.

Aminoglycoside acetyltransferases (AACs) catalyze the transfer of an acetyl group between acetyl-CoA and an aminoglycoside, producing CoA and an acetylated aminoglycoside. AAC(6′)-Ii enzymes target the amino group linked to the 6′ C atom in an aminoglycoside. Several structures of the AAC(6′)-Ii from Enterococcus faecium [Ef-AAC(6′)-Ii] have been reported to date. However, the detailed mechanism of its enzymatic function remains elusive. In this study, the crystal structure of Ef-AAC(6′)-Ii was determined in a novel substrate-free form. Based on structural analysis, it is proposed that Ef-AAC(6′)-Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef-AAC(6′)-Ii.

Bacterial cytochrome P450 (CYP) enzymes are involved in the hydroxylation of various endogenous substrates while using a heme molecule as a cofactor. CYPs have gained biotechnological interest as useful biocatalysts capable of altering chemical structures by adding a hydroxyl group in a regiospecific manner. Here, we identified, purified, and characterized two CYP154C4 proteins from Streptomyces sp. W2061 (StCYP154C4-1) and Streptomyces sp. ATCC 11861 (StCYP154C4-2). Activity assays showed that both StCYP154C4-1 and StCYP154C4-2 can produce 2'-hydroxylated testosterone, which differs from the activity of a previously described NfCYP154C5 from Nocardia farcinica in terms of its 16α-hydroxylation of testosterone. To better understand the molecular basis of the regioselectivity of these two CYP154C4 proteins, crystal structures of the ligand-unbound form of StCYP154C4-1 and the testosterone-bound form of StCYP154C4-2 were determined. Comparison with the previously determined NfCYP154C5 structure revealed differences in the substrate-binding residues, suggesting a likely explanation for the different patterns of testosterone hydroxylation, despite the high sequence similarities between the enzymes (54% identity). These findings provide valuable insights that will enable protein engineering for the development of artificial steroid-related CYPs exhibiting different regiospecificity.

Background S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. Results A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. Conclusions Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure–function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH. Electronic supplementary material The online version of this article (10.1186/s12934-019-1190-1) contains supplementary material, which is available to authorized users.

In cold and harsh environments such as glaciers and sediments in ice cores, microbes can survive by forming spores. Spores are composed of a thick coat protein, which protects against external factors such as heat-shock, high salinity, and nutrient deficiency. GerE is a key transcription factor involved in spore coat protein expression in the mother cell during sporulation. GerE regulates transcription during the late sporulation stage by directly binding to the promoter of cotB gene. Here, we report the crystal structure of PaGerE at 2.09 A resolution from Paenisporosarcina sp. TG-14, which was isolated from the Taylor glacier. The PaGerE structure is composed of four α-helices and adopts a helix-turn-helix architecture with 68 amino acid residues. Based on our DNA binding analysis, the PaGerE binds to the promoter region of CotB to affect protein expression. Additionally, our structural comparison studies suggest that DNA binding by PaGerE causes a conformational change in the α4-helix region, which may strongly induce dimerization of PaGerE.

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (

Additional file 1: Fig. S1. Phylogenetic analysis of SfSFGH. Fig. S2. Gene clustering analysis of SfSFGH. Fig. S3. Recombinant SfSFGH protein purification, crystallization, and X-ray diffraction data collection. Fig. S4. Freezeâ thaw cycles of SfSFGH.

Curcumin is a yellow-colored ingredient in dietary spice turmeric ( Curcuma longa Linn). This nontoxic polyphenol has antitumor, anti-inflammatory, apoptotic, and antioxidant activities. The ingested curcumin is reduced to multihydrated forms with more potent therapeutic potentials by the curcumin reductase (CurA) from commensal Escherichia coli. In this study, we demonstrated that Vibrio vulnificus CurA ( VvCurA) with 87% sequence similarity to the E. coli CurA exhibits the curcumin-reducing activity through spectrophotometric detection of NADPH oxidation and high performance liquid chromatographic analysis of curcumin consumption and product generation. Afterward, we determined the crystal structures of VvCurA and the VvCurA/NADPH complex, and made the in silico model of the VvCurA/NADPH/curcumin ternary complex through induced fit docking. Based on structural information, active site residues that play critical roles in catalysis have been identified and characterized by mutational and kinetic studies, leading us to propose the reaction mechanism of CurA.

Cold-active acetyl xylan esterases allow for reduced bioreactor heating costs in bioenergy production. Here, we isolated and characterized a cold-active acetyl xylan esterase (PbAcE) from the psychrophilic soil microbe Paenibacillus sp. R4. The enzyme hydrolyzes glucose penta-acetate and xylan acetate, reversibly producing acetyl xylan from xylan, and it shows higher activity at 4°C than at 25°C. We solved the crystal structure of PbAcE at 2.1-A resolution to investigate its active site and the reason for its low-temperature activity. Structural analysis showed that PbAcE forms a hexamer with a central substrate binding tunnel, and the inter-subunit interactions are relatively weak compared with those of its mesophilic and thermophilic homologs. PbAcE also has a shorter loop and different residue composition in the β4–α3 and β5–α4 regions near the substrate binding site. Flexible subunit movements and different active site loop conformations may enable the strong low-temperature activity and broad substrate specificity of PbAcE. In addition, PbAcE was found to have strong activity against antibiotic compound substrates, such as cefotaxime and 7-amino cephalosporanic acid (7-ACA). In conclusion, the PbAcE structure and our biochemical results provide the first example of a cold-active acetyl xylan esterase and a starting template for structure-based protein engineering.

Endo-β-1,4-d-mannanase from the Antarctic springtail, Cryptopygus antarcticus (CaMan), is a cold-adapted β-mannanase that has the lowest optimum temperature (30°C) of all known β-mannanases. Here, we report the apo- and mannopentaose (M5) complex structures of CaMan. Structural comparison of CaMan with other β-mannanases from the multicellular animals reveals that CaMan has an extended loop that alters topography of the active site. Structural and mutational analyses suggest that this extended loop is linked to the cold-adapted enzymatic activity. From the CaMan-M5 complex structure, we defined the mannose-recognition subsites and observed unreported M5 binding site on the surface of CaMan. Proteins 2014; 82:3217–3223. © 2014 Wiley Periodicals, Inc.

MurF adds d -Ala- d -Ala dipeptide to UDP-N-acetylmuramyl- l -Ala-γ- d -Glu-m-DAP (or l -Lys) in an ATP-dependent manner, which is the last step in the biosynthesis of monomeric precursor of peptidoglycan. Here we report crystal structures of two MurF–ATP complexes: the MurF–ATP complex and the MurF–ATP–UDP complex. The ATP-binding mode revealed by the crystal structure of the MurF–ATP complex confirms the previous biochemical demonstration that a carbamoylated lysine and two Mg2+ ions are required for enzyme activity of MurF. The UDP–MurF interactions observed in the crystal structure of the MurF–ATP–UDP complex depict the characteristic substrate-binding mode of MurF. The emergence and dissemination of multidrug-resistant Acinetobacter baumannii strains are great threats to public health. Therefore, the structural information on A. baumannii MurF as a validated target for drug discovery will provide a framework to develop antibacterial agents against multidrug-resistant A. baumannii infections as well as to understand the reaction mechanism of MurF.

The CaMan gene product from Cryptopygus antarcticus, which belongs to the glycoside hydrolase family 5 type β-1,4-D-mannanases, has been crystallized using a precipitant solution consisting of 0.1 M Tris-HCl pH 8.5, 25%(w/v) polyethylene glycol 3350 by the microbatch crystallization method at 295 K. The CaMan protein crystal belonged to space group P212121, with unit-cell parameters a = 73.40, b = 83.81, c = 163.55 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 61.29%. CaMan-mannopentaose (M5) complex crystals that were isomorphous to the CaMan crystals were obtained using the same mother liquor containing 1 mM M5.

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a β-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β-lactam antibiotics. This result explains the weak β-lactamase activity of EstU1 compared with class C β-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum. Proteins 2013; 81:2045–2051. © 2013 Wiley Periodicals, Inc.

The yncB gene product from Vibrio vulnificus, which belongs to the medium-chain dehydrogenase/reductase (MDR) superfamily, was crystallized using the microbatch crystallization method at 295 K. Diffraction data sets were collected using synchrotron radiation. Crystals of selenomethionine-substituted YncB protein belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 90.52, b = 91.56, c = 104.79 Å. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 57%. Crystals of the YncB-NADP(H) complex belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 90.14, c = 105.61 Å. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 56.42%.

The surface of proteins can be charged with zinc ions and the anomalous signals from these zinc ions can be used for structure determination of proteins., Zinc is a suitable metal for anomalous dispersion phasing methods in protein crystallography. Structure determination using zinc anomalous scattering has been almost exclusively limited to proteins with intrinsically bound zinc(s). Here, it is reported that multiple zinc ions can easily be charged onto the surface of proteins with no intrinsic zinc-binding site by using zinc-containing solutions. Zn derivatization of protein surfaces appears to be a largely unnoticed but promising method of protein structure determination.

Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinson's disease is discussed.

The interaction between fermentation-respiration switch (FrsA) protein and glucose-specific enzyme IIA(Glc) increases glucose fermentation under oxygen-limited conditions. We show that FrsA converts pyruvate to acetaldehyde and carbon dioxide in a cofactor-independent manner and that its pyruvate decarboxylation activity is enhanced by the dephosphorylated form of IIA(Glc) (d-IIA(Glc)). Crystal structures of FrsA and its complex with d-IIA(Glc) revealed residues required for catalysis as well as the structural basis for the activation by d-IIA(Glc).

The emergence and global spread of multidrug-resistantAcinetobacter baumanniistrains are major threats to public health. Inhibition of peptidoglycan biosynthesis is an effective strategy for the development of antibiotics. The ATP-dependent UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) that is responsible for the last step of peptidoglycan biosynthesis is a validated target for the development of antibiotics. Crystals ofA. baumanniiMurF in complex with ATP were grown by the microbatch crystallization method at 295 K. The crystals belonged to space groupP3221, with unit-cell parametersa=b= 85.42,c= 129.86 Å. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 54.32%.

Endo-β-1,4-D-mannanase from the Antarctic springtail, Cryptopygus antarcticus (CaMan), is a cold-adapted β-mannanase that has the lowest optimum temperature (30°C) of all known β-mannanases. Here, we report the apo- and mannopentaose (M5) complex structures of CaMan. Structural comparison of CaMan with other β-mannanases from the multicellular animals reveals that CaMan has an extended loop that alters topography of the active site. Structural and mutational analyses suggest that this extended loop is linked to the cold-adapted enzymatic activity. From the CaMan-M5 complex structure, we defined the mannose-recognition subsites and observed unreported M5 binding site on the surface of CaMan.

De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu Kα X-ray source. The successful in-house Zn-SAD phasing of glucose isomerase, based on the anomalous signals of both zinc ions introduced to crystals by soaking and native sulfur atoms, drove us to determine the structure of SaFur, a zinc-containing transcription factor, by Zn-SAD phasing using in-house X-ray source. The abundance of zinc-containing proteins in nature, the easy zinc derivatization of the protein surface, no need of synchrotron access, and the successful experimental phasing with the modest 2.6 Å resolution SAD data indicate that in-house Zn-SAD phasing can be widely applicable to structure determination.

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a β-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β-lactam antibiotics. This result explains the weak β-lactamase activity of EstU1 compared with class C β-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum.