Pub Date : 2015-12-01DOI: 10.1107/S1399004715018635
M. Ferraroni, S. del Prete, D. Vullo, C. Capasso, C. Supuran
Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate (hydrogen carbonate) and a proton. CAs have been extensively investigated owing to their involvement in numerous physiological and pathological processes. Currently, CA inhibitors are widely used as antiglaucoma, anticancer and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi and bacteria has emerged as a new research direction. In this article, the cloning and kinetic characterization of the β-CA from Vibrio cholerae (VchCAβ) are reported. The X-ray crystal structure of this new enzyme was solved at 1.9 Å resolution from a crystal that was perfectly merohedrally twinned, revealing a tetrameric type II β-CA with a closed active site in which the zinc is tetrahedrally coordinated to Cys42, Asp44, His98 and Cys101. The substrate bicarbonate was found bound in a noncatalytic binding pocket close to the zinc ion, as reported for a few other β-CAs, such as those from Escherichia coli and Haemophilus influenzae. At pH 8.3, the enzyme showed a significant catalytic activity for the physiological reaction of the hydration of CO2 to bicarbonate and protons, with the following kinetic parameters: a kcat of 3.34 × 10(5) s(-1) and a kcat/Km of 4.1 × 10(7) M(-1) s(-1). The new enzyme, on the other hand, was poorly inhibited by acetazolamide (Ki of 4.5 µM). As this bacterial pathogen encodes at least three CAs, an α-CA, a β-CA and a γ-CA, these enzymes probably play an important role in the life cycle and pathogenicity of Vibrio, and it cannot be excluded that interference with their activity may be exploited therapeutically to obtain antibiotics with a different mechanism of action.
{"title":"Crystal structure and kinetic studies of a tetrameric type II β-carbonic anhydrase from the pathogenic bacterium Vibrio cholerae.","authors":"M. Ferraroni, S. del Prete, D. Vullo, C. Capasso, C. Supuran","doi":"10.1107/S1399004715018635","DOIUrl":"https://doi.org/10.1107/S1399004715018635","url":null,"abstract":"Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate (hydrogen carbonate) and a proton. CAs have been extensively investigated owing to their involvement in numerous physiological and pathological processes. Currently, CA inhibitors are widely used as antiglaucoma, anticancer and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi and bacteria has emerged as a new research direction. In this article, the cloning and kinetic characterization of the β-CA from Vibrio cholerae (VchCAβ) are reported. The X-ray crystal structure of this new enzyme was solved at 1.9 Å resolution from a crystal that was perfectly merohedrally twinned, revealing a tetrameric type II β-CA with a closed active site in which the zinc is tetrahedrally coordinated to Cys42, Asp44, His98 and Cys101. The substrate bicarbonate was found bound in a noncatalytic binding pocket close to the zinc ion, as reported for a few other β-CAs, such as those from Escherichia coli and Haemophilus influenzae. At pH 8.3, the enzyme showed a significant catalytic activity for the physiological reaction of the hydration of CO2 to bicarbonate and protons, with the following kinetic parameters: a kcat of 3.34 × 10(5) s(-1) and a kcat/Km of 4.1 × 10(7) M(-1) s(-1). The new enzyme, on the other hand, was poorly inhibited by acetazolamide (Ki of 4.5 µM). As this bacterial pathogen encodes at least three CAs, an α-CA, a β-CA and a γ-CA, these enzymes probably play an important role in the life cycle and pathogenicity of Vibrio, and it cannot be excluded that interference with their activity may be exploited therapeutically to obtain antibiotics with a different mechanism of action.","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78032550","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01DOI: 10.1107/S1399004715019331
N. Janardan, R. Harijan, T. Kiema, R. Wierenga, M. Murthy
Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) from Mycobacterium smegmatis in apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domain via flexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The Lα1-covering loop-Lα2 region, together with the Nβ2-Nα2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains.
硫硫酶催化3-酮酰基辅酶a分子的降解和合成。本文描述了耻垢分枝杆菌中载脂蛋白和配体形式的t1样硫酶(MSM-13硫酶)的晶体结构。系统比较了三种不同晶体形式的六种晶体学独立的非配体MSM-13硫硫酶四聚体(紧密二聚体的二聚体)，发现这两种紧密二聚体通过柔性铰链区域连接到刚性四聚结构域，产生不对称四聚体。在配体结构中，辅酶a与那些向相反二聚体的四聚环顶端旋转的亚基结合，表明该环对底物结合很重要。负责这种旋转的铰链区域位于Val123和Arg149附近。覆盖l α1的环- l α2区与相邻亚基的Nβ2-Nα2环形成了一个比其他四聚硫酶更大、极性更强的特异性口袋，表明MSM-13硫酶具有明显的底物特异性。与这一发现一致的是，在降解方向上，仅以乙酰辅酶a为底物检测到残留活性。乙酰辅酶a在合成方向上无活性。与其他已知巯基酶的结构比较表明，MSM-13巯基酶可能是一种降解巯基酶，对3-酮酰基辅酶a分子具有特异性，具有极性，大的酰基链。
{"title":"Structural characterization of a mitochondrial 3-ketoacyl-CoA (T1)-like thiolase from Mycobacterium smegmatis.","authors":"N. Janardan, R. Harijan, T. Kiema, R. Wierenga, M. Murthy","doi":"10.1107/S1399004715019331","DOIUrl":"https://doi.org/10.1107/S1399004715019331","url":null,"abstract":"Thiolases catalyze the degradation and synthesis of 3-ketoacyl-CoA molecules. Here, the crystal structures of a T1-like thiolase (MSM-13 thiolase) from Mycobacterium smegmatis in apo and liganded forms are described. Systematic comparisons of six crystallographically independent unliganded MSM-13 thiolase tetramers (dimers of tight dimers) from three different crystal forms revealed that the two tight dimers are connected to a rigid tetramerization domain via flexible hinge regions, generating an asymmetric tetramer. In the liganded structure, CoA is bound to those subunits that are rotated towards the tip of the tetramerization loop of the opposing dimer, suggesting that this loop is important for substrate binding. The hinge regions responsible for this rotation occur near Val123 and Arg149. The Lα1-covering loop-Lα2 region, together with the Nβ2-Nα2 loop of the adjacent subunit, defines a specificity pocket that is larger and more polar than those of other tetrameric thiolases, suggesting that MSM-13 thiolase has a distinct substrate specificity. Consistent with this finding, only residual activity was detected with acetoacetyl-CoA as the substrate in the degradative direction. No activity was observed with acetyl-CoA in the synthetic direction. Structural comparisons with other well characterized thiolases suggest that MSM-13 thiolase is probably a degradative thiolase that is specific for 3-ketoacyl-CoA molecules with polar, bulky acyl chains.","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88224061","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01DOI: 10.1107/S139900471501799X
K. Green, N. Qasim, Garik Gdaelvsky, Anna Kogan, Y. Goldgur, A. Parola, O. Lotan, O. Almog
Tryptophanase (Trpase) is a pyridoxal 5'-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein-protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one group, residues 15 and 59 that are located along the molecular axis R (also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (also termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.
{"title":"A structural view of the dissociation of Escherichia coli tryptophanase.","authors":"K. Green, N. Qasim, Garik Gdaelvsky, Anna Kogan, Y. Goldgur, A. Parola, O. Lotan, O. Almog","doi":"10.1107/S139900471501799X","DOIUrl":"https://doi.org/10.1107/S139900471501799X","url":null,"abstract":"Tryptophanase (Trpase) is a pyridoxal 5'-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein-protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one group, residues 15 and 59 that are located along the molecular axis R (also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (also termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"74798056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01DOI: 10.1107/S1399004715019598
M. Saoji, P. Paukstelis
DNA has proved to be a remarkable molecule for the construction of sophisticated two-dimensional and three-dimensional architectures because of its programmability and structural predictability provided by complementary Watson-Crick base pairing. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. This study examines how a single-nucleotide addition to a class of self-assembling DNA 13-mers leads to a significantly different overall structure under identical crystallization conditions. The DNA 13-mers self-assemble in the presence of Mg(2+) through a combination of Watson-Crick and noncanonical base-pairing interactions. The crystal structures described here show that all of the predicted Watson-Crick base pairs are present, with the major difference being a significant rearrangement of noncanonical base pairs. This includes the formation of a sheared A-G base pair, a junction of strands formed from base-triple interactions, and tertiary interactions that generate structural features similar to tandem sheared G-A base pairs. The adoption of this alternate noncanonical structure is dependent in part on the sequence in the Watson-Crick duplex region. These results provide important new insights into the sequence-structure relationship of short DNA oligonucleotides and demonstrate a unique interplay between Watson-Crick and noncanonical base pairs that is responsible for crystallization fate.
{"title":"Sequence-dependent structural changes in a self-assembling DNA oligonucleotide.","authors":"M. Saoji, P. Paukstelis","doi":"10.1107/S1399004715019598","DOIUrl":"https://doi.org/10.1107/S1399004715019598","url":null,"abstract":"DNA has proved to be a remarkable molecule for the construction of sophisticated two-dimensional and three-dimensional architectures because of its programmability and structural predictability provided by complementary Watson-Crick base pairing. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. This study examines how a single-nucleotide addition to a class of self-assembling DNA 13-mers leads to a significantly different overall structure under identical crystallization conditions. The DNA 13-mers self-assemble in the presence of Mg(2+) through a combination of Watson-Crick and noncanonical base-pairing interactions. The crystal structures described here show that all of the predicted Watson-Crick base pairs are present, with the major difference being a significant rearrangement of noncanonical base pairs. This includes the formation of a sheared A-G base pair, a junction of strands formed from base-triple interactions, and tertiary interactions that generate structural features similar to tandem sheared G-A base pairs. The adoption of this alternate noncanonical structure is dependent in part on the sequence in the Watson-Crick duplex region. These results provide important new insights into the sequence-structure relationship of short DNA oligonucleotides and demonstrate a unique interplay between Watson-Crick and noncanonical base pairs that is responsible for crystallization fate.","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"89944400","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01DOI: 10.1107/S139900471501603X
Zhang Tao, De-qiang Yao, Jiawei Wang, Yuan-xin Gu, H. Fan
A post-experimental identification/purification procedure similar to that described in Zhang et al. [(2015), IUCrJ, 2, 322-326] has been proposed for use in the treatment of multiphase protein serial crystallography (SX) diffraction snapshots. As a proof of concept, the procedure was tested using theoretical serial femtosecond crystallography (SFX) data from a mixture containing native and derivatized crystals of a protein. Two known proteins were taken as examples. Multiphase diffraction snapshots were subjected to two rounds of indexing using the program CrystFEL [White et al. (2012). J. Appl. Cryst. 45, 335-341]. In the first round, an ab initio indexing was performed to derive a set of approximate primitive unit-cell parameters, which are roughly the average of those from the native protein and the derivative. These parameters were then used in a second round of indexing as input to CrystFEL. The results were then used to separate the diffraction snapshots into two subsets corresponding to the native and the derivative. For each test sample, integration of the two subsets of snapshots separately led to two sets of three-dimensional diffraction intensities, one belonging to the native and the other to the derivative. Based on these two sets of intensities, a conventional single isomorphous replacement (SIR) procedure solved the structure easily.
与Zhang等人[(2015)，IUCrJ, 2,322 -326]所描述的实验后鉴定/纯化程序类似，已被提出用于处理多相蛋白质序列晶体学(SX)衍射快照。作为概念证明，该程序使用理论序列飞秒晶体学(SFX)数据进行了测试，这些数据来自含有天然和衍生蛋白质晶体的混合物。以两种已知的蛋白质为例。使用CrystFEL程序对多相衍射快照进行了两轮索引[White et al.(2012)]。j:。[j].中国生物医学工程学报，2016,35(5):335-341。在第一轮中，进行从头开始索引以获得一组近似的原始单位细胞参数，这些参数大致是来自天然蛋白质和衍生物的平均值。然后在第二轮索引中使用这些参数作为输入到CrystFEL。然后利用结果将衍射快照分为对应于原生和导数的两个子集。对于每个测试样品，分别对两个快照子集进行积分，得到两组三维衍射强度，一组属于原生衍射强度，另一组属于导数衍射强度。基于这两组强度，传统的单同构替换(SIR)方法可以很容易地求解该结构。
{"title":"Serial crystallographic analysis of protein isomorphous replacement data from a mixture of native and derivative microcrystals.","authors":"Zhang Tao, De-qiang Yao, Jiawei Wang, Yuan-xin Gu, H. Fan","doi":"10.1107/S139900471501603X","DOIUrl":"https://doi.org/10.1107/S139900471501603X","url":null,"abstract":"A post-experimental identification/purification procedure similar to that described in Zhang et al. [(2015), IUCrJ, 2, 322-326] has been proposed for use in the treatment of multiphase protein serial crystallography (SX) diffraction snapshots. As a proof of concept, the procedure was tested using theoretical serial femtosecond crystallography (SFX) data from a mixture containing native and derivatized crystals of a protein. Two known proteins were taken as examples. Multiphase diffraction snapshots were subjected to two rounds of indexing using the program CrystFEL [White et al. (2012). J. Appl. Cryst. 45, 335-341]. In the first round, an ab initio indexing was performed to derive a set of approximate primitive unit-cell parameters, which are roughly the average of those from the native protein and the derivative. These parameters were then used in a second round of indexing as input to CrystFEL. The results were then used to separate the diffraction snapshots into two subsets corresponding to the native and the derivative. For each test sample, integration of the two subsets of snapshots separately led to two sets of three-dimensional diffraction intensities, one belonging to the native and the other to the derivative. Based on these two sets of intensities, a conventional single isomorphous replacement (SIR) procedure solved the structure easily.","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72979233","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01DOI: 10.1107/S1399004715018672
H. V. Solomon, Orly Tabachnikov, S. Lansky, R. Salama, H. Feinberg, Y. Shoham, G. Shoham
Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.
{"title":"Structure-function relationships in Gan42B, an intracellular GH42 β-galactosidase from Geobacillus stearothermophilus.","authors":"H. V. Solomon, Orly Tabachnikov, S. Lansky, R. Salama, H. Feinberg, Y. Shoham, G. Shoham","doi":"10.1107/S1399004715018672","DOIUrl":"https://doi.org/10.1107/S1399004715018672","url":null,"abstract":"Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73759356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01Epub Date: 2015-11-26DOI: 10.1107/S1399004715018702
Nicholas Skar-Gislinge, Søren A R Kynde, Ilia G Denisov, Xin Ye, Ivan Lenov, Stephen G Sligar, Lise Arleth
Membrane proteins reconstituted into phospholipid nanodiscs comprise a soluble entity accessible to solution small-angle X-ray scattering (SAXS) studies. It is demonstrated that using SAXS data it is possible to determine both the shape and localization of the membrane protein cytochrome P450 3A4 (CYP3A4) while it is embedded in the phospholipid bilayer of a nanodisc. In order to accomplish this, a hybrid approach to analysis of small-angle scattering data was developed which combines an analytical approach to describe the multi-contrast nanodisc with a free-form bead-model description of the embedded protein. The protein shape is then reconstructed ab initio to optimally fit the data. The result of using this approach is compared with the result obtained using a rigid-body description of the CYP3A4-in-nanodisc system. Here, the CYP3A4 structure relies on detailed information from crystallographic and molecular-dynamics studies of CYP3A4. Both modelling approaches arrive at very similar solutions in which the α-helical anchor of the CYP3A4 systematically stays close to the edge of the nanodisc and with the large catalytic domain leaning over the outer edge of the nanodisc. The obtained distance between the globular domains of CYP3A4 is consistent with previously published theoretical calculations.
{"title":"Small-angle scattering determination of the shape and localization of human cytochrome P450 embedded in a phospholipid nanodisc environment.","authors":"Nicholas Skar-Gislinge, Søren A R Kynde, Ilia G Denisov, Xin Ye, Ivan Lenov, Stephen G Sligar, Lise Arleth","doi":"10.1107/S1399004715018702","DOIUrl":"10.1107/S1399004715018702","url":null,"abstract":"<p><p>Membrane proteins reconstituted into phospholipid nanodiscs comprise a soluble entity accessible to solution small-angle X-ray scattering (SAXS) studies. It is demonstrated that using SAXS data it is possible to determine both the shape and localization of the membrane protein cytochrome P450 3A4 (CYP3A4) while it is embedded in the phospholipid bilayer of a nanodisc. In order to accomplish this, a hybrid approach to analysis of small-angle scattering data was developed which combines an analytical approach to describe the multi-contrast nanodisc with a free-form bead-model description of the embedded protein. The protein shape is then reconstructed ab initio to optimally fit the data. The result of using this approach is compared with the result obtained using a rigid-body description of the CYP3A4-in-nanodisc system. Here, the CYP3A4 structure relies on detailed information from crystallographic and molecular-dynamics studies of CYP3A4. Both modelling approaches arrive at very similar solutions in which the α-helical anchor of the CYP3A4 systematically stays close to the edge of the nanodisc and with the large catalytic domain leaning over the outer edge of the nanodisc. The obtained distance between the globular domains of CYP3A4 is consistent with previously published theoretical calculations.</p>","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667284/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85978420","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01DOI: 10.1107/S1399004715018520
Alessandra Del Giudice, N. Pavel, L. Galantini, G. Falini, P. Trost, S. Fermani, F. Sparla
Oxygenic photosynthetic organisms produce sugars through the Calvin-Benson cycle, a metabolism that is tightly linked to the light reactions of photosynthesis and is regulated by different mechanisms, including the formation of protein complexes. Two enzymes of the cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), form a supramolecular complex with the regulatory protein CP12 with the formula (GAPDH-CP122-PRK)2, in which both enzyme activities are transiently inhibited during the night. Small-angle X-ray scattering analysis performed on both the GAPDH-CP12-PRK complex and its components, GAPDH-CP12 and PRK, from Arabidopsis thaliana showed that (i) PRK has an elongated, bent and screwed shape, (ii) the oxidized N-terminal region of CP12 that is not embedded in the GAPDH-CP12 complex prefers a compact conformation and (iii) the interaction of PRK with the N-terminal region of CP12 favours the approach of two GAPDH tetramers. The interaction between the GAPDH tetramers may contribute to the overall stabilization of the GAPDH-CP12-PRK complex, the structure of which is presented here for the first time.
{"title":"Unravelling the shape and structural assembly of the photosynthetic GAPDH-CP12-PRK complex from Arabidopsis thaliana by small-angle X-ray scattering analysis.","authors":"Alessandra Del Giudice, N. Pavel, L. Galantini, G. Falini, P. Trost, S. Fermani, F. Sparla","doi":"10.1107/S1399004715018520","DOIUrl":"https://doi.org/10.1107/S1399004715018520","url":null,"abstract":"Oxygenic photosynthetic organisms produce sugars through the Calvin-Benson cycle, a metabolism that is tightly linked to the light reactions of photosynthesis and is regulated by different mechanisms, including the formation of protein complexes. Two enzymes of the cycle, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK), form a supramolecular complex with the regulatory protein CP12 with the formula (GAPDH-CP122-PRK)2, in which both enzyme activities are transiently inhibited during the night. Small-angle X-ray scattering analysis performed on both the GAPDH-CP12-PRK complex and its components, GAPDH-CP12 and PRK, from Arabidopsis thaliana showed that (i) PRK has an elongated, bent and screwed shape, (ii) the oxidized N-terminal region of CP12 that is not embedded in the GAPDH-CP12 complex prefers a compact conformation and (iii) the interaction of PRK with the N-terminal region of CP12 favours the approach of two GAPDH tetramers. The interaction between the GAPDH tetramers may contribute to the overall stabilization of the GAPDH-CP12-PRK complex, the structure of which is presented here for the first time.","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"91071561","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01DOI: 10.1107/S1399004715018659
T. Manszewski, Kriti Singh, B. Imiolczyk, M. Jaskólski
S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM, S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase from Bradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 Å resolution, showing the domain organization (substrate-binding domain, NAD(+) cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.
s -腺苷- l-同型半胱氨酸水解酶(SAHase)参与s -腺苷- l-蛋氨酸(SAM)依赖性甲基化反应的酶促调节。在SAM的甲基转移后，s -腺苷- l-同型半胱氨酸(SAH)作为副产物形成，SAH又被SAHase水解成腺苷(Ado)和同型半胱氨酸(Hcy)。豆科植物固氮共生细菌elkanii慢生根瘤菌(Bradyrhizobium elkanii)的SAHase BeSAHase的晶体结构以1.7 Å分辨率测定，显示了亚基的结构域组织(底物结合域、NAD(+)辅因子结合域和二聚化域)。这种蛋白质以其生物学上相关的四聚体形式结晶，有三个亚基在一个封闭的构象中，与酶促反应的Ado产物形成复合物。第四个亚基是无配体的，具有开放的构象。因此，BeSAHase结构提供了由其天然配体结合诱导的酶的结构域运动的独特快照。
{"title":"An enzyme captured in two conformational states: crystal structure of S-adenosyl-L-homocysteine hydrolase from Bradyrhizobium elkanii.","authors":"T. Manszewski, Kriti Singh, B. Imiolczyk, M. Jaskólski","doi":"10.1107/S1399004715018659","DOIUrl":"https://doi.org/10.1107/S1399004715018659","url":null,"abstract":"S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM, S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase from Bradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 Å resolution, showing the domain organization (substrate-binding domain, NAD(+) cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1107/S1399004715018659","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72513339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2015-12-01Epub Date: 2015-11-26DOI: 10.1107/S1399004715018830
Yunfei Wu, Chengliang Wang, Shenglong Lin, Minhao Wu, Lu Han, Changlin Tian, Xuan Zhang, Jianye Zang
Staphylococcus aureus is a Gram-positive bacterium with strong pathogenicity that causes a wide range of infections and diseases. Enolase is an evolutionarily conserved enzyme that plays a key role in energy production through glycolysis. Additionally, enolase is located on the surface of S. aureus and is involved in processes leading to infection. Here, crystal structures of Sa_enolase with and without bound phosphoenolpyruvate (PEP) are presented at 1.6 and 2.45 Å resolution, respectively. The structure reveals an octameric arrangement; however, both dimeric and octameric conformations were observed in solution. Furthermore, enzyme-activity assays show that only the octameric variant is catalytically active. Biochemical and structural studies indicate that the octameric form of Sa_enolase is enzymatically active in vitro and likely also in vivo, while the dimeric form is catalytically inactive and may be involved in other biological processes.
{"title":"Octameric structure of Staphylococcus aureus enolase in complex with phosphoenolpyruvate.","authors":"Yunfei Wu, Chengliang Wang, Shenglong Lin, Minhao Wu, Lu Han, Changlin Tian, Xuan Zhang, Jianye Zang","doi":"10.1107/S1399004715018830","DOIUrl":"10.1107/S1399004715018830","url":null,"abstract":"<p><p>Staphylococcus aureus is a Gram-positive bacterium with strong pathogenicity that causes a wide range of infections and diseases. Enolase is an evolutionarily conserved enzyme that plays a key role in energy production through glycolysis. Additionally, enolase is located on the surface of S. aureus and is involved in processes leading to infection. Here, crystal structures of Sa_enolase with and without bound phosphoenolpyruvate (PEP) are presented at 1.6 and 2.45 Å resolution, respectively. The structure reveals an octameric arrangement; however, both dimeric and octameric conformations were observed in solution. Furthermore, enzyme-activity assays show that only the octameric variant is catalytically active. Biochemical and structural studies indicate that the octameric form of Sa_enolase is enzymatically active in vitro and likely also in vivo, while the dimeric form is catalytically inactive and may be involved in other biological processes.</p>","PeriodicalId":6895,"journal":{"name":"Acta Crystallographica Section D: Biological Crystallography","volume":null,"pages":null},"PeriodicalIF":2.2,"publicationDate":"2015-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1107/S1399004715018830","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"61948588","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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