Crystal Structure of a Curcumin-converting Enzyme Homologue, YncB, from Vibrio vulnificus

비브리오 불니피쿠스에 존재하는 YncB 단백질은 MDR 계열의 NADPH 의존적 산화환원 효소이다. 수 많은 MDR 계열 단백질이 모든 생물종에 존재하지만 아직 그 생물학적 기능은 많이 알려져 있지 않다. 최근 대장균에서 발견된 MDR 계열의 CurA 단백질은 의약학 분야에서 광범위하게 효능이 알려진 curcumin을 활성이 높은 형태로 전환하는 효소로서 유사 단백질이 다양한 병원성 미생물에서 발견되고 있다. Curcumin 전환 기작의 정확한 이해를 위해 해양 미생물인 비브리오 불니피쿠스에 존재하는 CurA 유사 단백질인 YncB의 3차원 결정 구조를 규명하였다.약학 분야에서 광범위하게 효능이 알려진 curcumin을 활성이 높은 형태로 전환하는 효소로서 유사 단백질이 다양한 병원성 미생물에서 발견되고 있다. Curcumin 전환 기작의 정확한 이해를 위해 해양 미생물인 비브리오 불니피쿠스에 존재하는 CurA 유사 단백질인 YncB의 3차원 결정 구조를 규명하였다.2

Cultivation at 6-10 is Effective to Overcome the Insolubility of Recombinant Proteins

Protein expression in Escherichia coli at 15–25 ℃ is widely used to increase the solubility of recombinant proteins. However, many recombinant proteins are insolubly expressed even at those low temperatures. Here, we show that recombinant proteins can be expressed as soluble forms by simply lowering temperature to 6–10 ℃ without cold adapted chaperon systems. By using E. coli Rosetta-gami2 (DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 l culture grown at 6 and 10 ℃, respectively. Cultivation at 10 ℃ also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21 (DE3). Consequently, E. coli cultivation at 6–10 ℃ is an effective strategy for overcoming a major hurdle of the inclusion body formation. Mannanase (CaMan) and cellulase (CaCel) from Cryptopygus antarticus was purified and crystallized at 295 K. A 2.6 Å resolution data set of mannanase crystal has been collected using synchrotron radiation. The crystals belong to space group P212121 with unit-cell parameters a = 74.02, b = 82.45, c = 164.32 Å. A 2.6 Å resolution data set of cellulase crystal has been collected using synchrotron radiation. The crystals belong to space group P3121 with unit-cell parameters a = 81.71, b = 81.71, c = 89.35 Å, alpa= 90,beta= 90, gamma= 12combinant proteins can be expressed as soluble forms by simply lowering temperature to 6–10 ℃ without cold adapted chaperon systems. By using E. coli Rosetta-gami2 (DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 l culture grown at 6 and 10 ℃, respectively. Cultivation at 10 ℃ also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21 (DE3). Consequently, E. coli cultivation at 6–10 ℃ is an effective strateg1

Crystal Structure of Filamentous Aggregates of Human DJ-1

Mutations in the DJ-1 gene have been implicated in the autosomal recessive early onset parkinsonism. DJ-1 is a soluble dimeric protein with critical roles in response to oxidative stress and in neuronal maintenance. However, several lines of evidence suggest the existence of a nonfunctional aggregated form of DJ-1 in the brain of patients with some neurodegenerative diseases. Here, we show that inorganic phosphate, an important anion that exhibits elevated levels in patients with Parkinson disease, transforms DJ-1 into filamentous aggregates. According to the 2.4-Å crystal structure, DJ-1 dimers are linearly stacked through Pi-mediated interactions to form protofilaments, which are then bundled into a filamentous assembly.es of evidence suggest the existence of a nonfunctional aggregated form of DJ-1 in the brain of patients with some neurodegenerative diseases. Here, we show that inorganic phosphate, an important anion that exhibits elevated levels in patients with Parkinson disease, transforms DJ-1 into filamentous aggregates. According to the 2.4-Å crystal structure, DJ-1 dimers are linearly stacked through Pi-mediated interactions to form protofilaments, which are then bundled into a filamentous assembly.1

Crystal structures of a cold-active β-mannanase and its mannopentaose complex from Antarctic springtail, Cryptopygus antarcticus

The freezing-intolerant Antarctic springtail, Cryptopygus antarcticus Willem, is the most abundant and widespread terrestrial micro-arthropod in the maritime Antarctic region. The adult reaches approximately 1.2 mm in body length with a live weight of 55 - 78 μg, containing 69 - 85 % water. This organism feeds on fungi, unicellular algae and detritus. Therefore, it is thought that C. antarcticus contains various kinds of cold-active enzymes that hydrolyze carbohydrates. From expressed sequence tags (ESTs) library of C. antarcticus, we identified the β-1,4-D-mannanase gene (CaMan) that belongs to GH 5 family. CaMan is a 382-residue protein with a putative signal peptide and exhibits high specific activity toward locust bean gum at an optimal temperature of 303 K and an optimal pH of 3.5. Its optimal temperature is the lowes among those of the known mannanases and the optimal pH is also the lowest except for those of Sclerotium rolfsii and Aspergillus sulphurous enzymes (pH 2.9 and pH 2.4, respectively). Even at 273 - 278 K, CaMan retains 20 - 40 % of its maximum activity. And it shows typical features of a cold-active enzymes which has a high frequency of polar residues such as Asn, Gln and Ser, and a low frequency of hydrophobic residues as well as a low ratio of Arg/(Arg+Lys) compared to the mesophilic β-mannanases. Here, we report the first structure of a GH5 β-mannanase in complex with M5 (CaMan/M5) and presene weight of 55 - 78 μg, containing 69 - 85 % water. This organism feeds on fungi, unicellular algae and detritus. Therefore, it is thought that C. antarcticus contains various kinds of cold-active enzymes that hydrolyze carbohydrates. From expressed sequence tags (ESTs) library of C. antarcticus, we identified the β-1,4-D-mannanase gene (CaMan) that belongs to GH 5 family. CaMan is a 382-residue protein with a putative signal peptide and exhibits high specific activity toward locust bean gum at an optimal temperature of 303 K and an optimal pH of 3.5. Its optimal temperature is the lowes among those of the known mannanases and the optimal pH is also the lowest except for those of Sclerotium rolfsii and Aspergillus sulphurous enzymes (pH 2.9 and pH 2.4, respectively). Even at 273 - 278 K, CaMan retains 20 - 40 % of its maximum activity. And it shows typical features of a cold-active enzymes which has a high frequency of polar residues such as Asn, Gln and Ser, and a low frequency of hydrophobic residues as well as a low ratio of Arg/(Arg+Lys) compared to the mesophilic β-mannanases. Here, we report the first structure of a GH5 β-mannanase in complex with M5 (CaMan/M5) and presen2

Cultivation at 6-10 ℃ is Effective to Overcome the Insolubility of Recombinant Proteins.

Protein expression in Escherichia coli at 15–25 ℃ is widely used to increase the solubility of recombinant proteins. However, many recombinant proteins are insolubly expressed even at those low temperatures. Here, we show that recombinant proteins can be expressed as soluble forms by simply lowering temperature to 6–10 ℃ without cold adapted chaperon systems. By using E. coli Rosetta-gami2 (DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 L culture grown at 6 and 10 ℃, respectively. Cultivation at 10 ℃ also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21 (DE3). Consequently, E. coli cultivation at 6–10 ℃ is an effective strategy for overcoming a major hurdle of the inclusion body formation. Mannanase (CaMan) and cellulase (CaCel) from Cryptopygus antarticus was purified and crystallized at 295 K. A 2.6 Å resolution data set of mannanase crystal has been collected using synchrotron radiation. The crystals belong to space group P212121 with unit-cell parameters a = 74.02, b = 82.45, c = 164.32 Å. A 2.6 Å resolution data set of cellulase crystal has been collected using synchrotron radiation. The crystals belong to space group P3121 with unit-cell parameters a = 81.71, b = 81.71, c = 89.35 Å, a= b = 90, γcombinant proteins can be expressed as soluble forms by simply lowering temperature to 6–10 ℃ without cold adapted chaperon systems. By using E. coli Rosetta-gami2 (DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 L culture grown at 6 and 10 ℃, respectively. Cultivation at 10 ℃ also led to successful expression of EM3L7 (a lipase isolated from a metagenomic library) in a soluble form in E. coli BL21 (DE3). Consequently, E. coli cultivation at 6–10 ℃ is an effective strateg2

Crystal structure of Lon protease

Lon proteases are distributed in all kingdoms of life and are required for survival of cells under stress. Lon is a tandem fusion of an AAA+ molecular chaperone and a protease with a serine-lysine catalytic dyad. We report the 2.0-Å resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three-tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight- and weak-binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP-driven protein unfolding and translocation. The bowl-shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.g; resolution crystal structure of Thermococcus onnurineus NA1 Lon (TonLon). The structure is a three-tiered hexagonal cylinder with a large sequestered chamber accessible through an axial channel. Conserved loops extending from the AAA+ domain combine with an insertion domain containing the membrane anchor to form an apical domain that serves as a gate governing substrate access to an internal unfolding and degradation chamber. Alternating AAA+ domains are in tight- and weak-binding nucleotide states with different domain orientations and intersubunit contacts, reflecting intramolecular dynamics during ATP-driven protein unfolding and translocation. The bowl-shaped proteolytic chamber is contiguous with the chaperone chamber allowing internalized proteins direct access to the proteolytic sites without further gating restrictions.1

Zinc-binding to protein surface and its application to phase determination.

Zinc is a suitable metal for anomalous dispersion phasing methods. Crystal structure determination using anomalous scattering from zinc has been limited to proteins with intrinsically bound zinc(s). Herein we report that multiple zinc ions can be charged on the surface of proteins and used for structure determination of proteins with no intrinsic zinc-binding site. The discovery of facile zinc-binding to proteins without an intrinsic zinc-binding site opens a new avenue to the usage of zinc anomalous signal for structure determination.ons can be charged on the surface of proteins and used for structure determination of proteins with no intrinsic zinc-binding site. The discovery of facile zinc-binding to proteins without an intrinsic zinc-binding site opens a new avenue to the usage of zinc anomalous signal for structure determination.2

Zinc-binding to protein surface and its application to phase determination.

Zinc is a suitable metal for anomalous dispersion phasing methods. Crystal structure determination using anomalous scattering from zinc has been limited to proteins with intrinsically bound zinc(s). Herein we report that multiple zinc ions can be charged on the surface of proteins and used for structure determination of proteins with no intrinsic zinc-binding site. The discovery of facile zinc-binding to proteins without an intrinsic zinc-binding site opens a new avenue to the usage of zinc anomalous signal for structure determination.ons can be charged on the surface of proteins and used for structure determination of proteins with no intrinsic zinc-binding site. The discovery of facile zinc-binding to proteins without an intrinsic zinc-binding site opens a new avenue to the usage of zinc anomalous signal for structure determination.1

Structural analysis of Nickel uptake regulator (Nur) and Zinc uptake regulator (Zur) from Streptomyces coelicolor

Various transition metal ions are essentially required for the cell growth and survival of almost all organisms. However, excess or lack of transition metal ions are very harmfulness. Thus, cells exploit highly sensitive metal ion-binding regulators to achieve homeostasis. In the present study, we describe the Crystal structure of (Nickel uptake regulator) Nur and (Zinc uptake regulator) Zur from Streptomyces coelicolor at 2.4-Å resolution. These two enzyme belongs to the ferric uptake regulator (Fur) family and controls expression of genes in response to available nickel or zinc. First, Nur is a nickel responsive transcription factor that controls nickel homeostasis and antioxidative response. It contains a unique nickel-specific metal site in addition to a nonspecific common metal site. The identification of the 6-5-6 motif of the Nur recognition box and a Nur/DNA complex model reveals that Nur mainly interacts with terminal bases of the palindrome on complex formation. This contrasts with more distributed contacts between Fur and the n-1-n type of the Fur-binding motif. seconds, Zur is zinc responsive transcription factor containing zinc-binding sites (C-,M- and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C- and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mng regulators to achieve homeostasis. In the present study, we describe the Crystal structure of (Nickel uptake regulator) Nur and (Zinc uptake regulator) Zur from Streptomyces coelicolor at 2.4-Å resolution. These two enzyme belongs to the ferric uptake regulator (Fur) family and controls expression of genes in response to available nickel or zinc. First, Nur is a nickel responsive transcription factor that controls nickel homeostasis and antioxidative response. It contains a unique nickel-specific metal site in addition to a nonspecific c1

Structural basis for the beta-lactamase activity of EstU1, a family VIII carboxylesterase

EstU1 is a remarkable family VIII carboxylesterase in that it exhibits hydrolytic activity towards the amide bond of clinically-used b-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. The crystal structures of EstU1 and the EstU1/cephalothin complex revealed the structural basis for the catalytic feature. Structural and sequential comparison of EstU1 with other family VIII carboxylesterases that cannot cleave clinically-used b-lactam antibiotics provides insights into an operative molecular strategy of family VIII carboxylesterases to extend their substrate spectrum. the EstU1/cephalothin complex revealed the structural basis for the catalytic feature. Structural and sequential comparison of EstU1 with other family VIII carboxylesterases that cannot cleave clinically-used b-lactam antibiotics provides insights into an operative molecular strategy of family VIII carboxylesterases to extend their substrate spectrum.2