From structure to function: insights into the catalytic substrate specificity and thermostability displayed by Bacillus subtilis mannanase BCman.

BCman, a beta-mannanase from the plant root beneficial bacterium Bacillus subtilis Z-2, has a potential to be used in the production of mannooligosaccharide, which shows defense induction activity on both melon and tobacco, and plays an important role in the biological control of plant disease. Here we report the biochemical properties and crystal structure of BCman-GH26 enzyme. Kinetic analysis reveals that BCman is an endo-beta-mannanase, specific for mannan, and has no activity on mannooligosaccharides. The catalytic acid/base Glu167 and nucleophile Glu266 are positioned on the beta4 and beta7 strands, respectively. The 1.45-A crystal structure reveals that BCman is a typical (beta/alpha)(8) folding type. One large difference from the saddle-shaped active center of other endo-beta-mannanases is the presence of a shallow-dish-shaped active center and substrate-binding site that are both unique to BCman. These differences are mainly due to important changes in the length and position of loop 1 (Phe37-Met47), loop 2 (Ser103-Ala134), loop3 (Phe162-Asn185), loop 4 (Tyr215-Ile236), loop 5 (Pro269-Tyr278), and loop 6 (Trp298-Gly309), all of which surround the active site. Data from isothermal titration calorimetry and crystallography indicated only two substrate-binding subsites (+1 and -1) within the active site of BCman. These two sites are involved in the enzyme's mannan degradation activity and in restricting the binding capacity for mannooligosaccharides. Binding and catalysis of BCman to mannan is mediated mainly by a surface containing a strip of solvent-exposed aromatic rings of Trp302, Trp298, Trp172, and Trp72. Additionally, BCman contains a disulfide bond (Cys66Cys86) and a special His1-His23-Glu336 metal-binding site. This secondary structure is a key factor in the enzyme's stability.

Crystal structure of human dynein light chain Dnlc2A: structural insights into the interaction with IC74.

The human light chain of the motor protein dynein, Dnlc2A, is also a novel TGF-beta-signaling component, which is altered with high frequency in epithelial ovarian cancer. It is an important mediator of dynein and the development of cancer, owing to its ability to bind to the dynein intermediate light chain (DIC) IC74 and to regulate TGF-beta-dependent transcriptional events. Here we report the 2.1-A crystal structure of Dnlc2A using single anomalous diffraction. The proteins form a homodimer in solution and interact mainly through the helix alpha(2), strand beta(3), and the loop following this strand in each protein to generate a 10-stranded beta-sheet core. The surface of the beta-sheet core is mainly positively charged and predicted (by software PPI-Pred) to be the site that interacts with other partners. At the same time, the residues 79-82, 88, and 90 of each molecule formed two holes in the core. Residue 89 of each molecule, which is crucial for the DIC binding function of Dnlc2A, is within the holes. On the basis of these observations, we propose that the homodimer is the structural and functional unit maintained by hydrogen bonding interactions and hydrophobic packing, and that the patch of the surface of the beta-sheet core is the main area of interaction with other partners. Furthermore, the two holes would be the key sites to interact with IC74.

Crystal structure of C-terminal desundecapeptide nitrite reductase from Achromobacter cycloclastes.

Monoclinic crystal structure of C-terminal desundecapeptide nitrite reductase (NiRc-11) from Achromobacter cycloclastes was determined at 2.6A. NiRc-11 exists as a loose trimer in the crystal. Deletion of 11 residues eliminates all intersubunit hydrogen bonds mediated by the C-terminal tail. The rigid irregular coil 105-112, which constitutes part of the sidewall of the active site pocket, undergoes conformational changes and becomes highly flexible in NiRc-11. Correspondingly, the linker segments between the two copper sites 95-100 and 135-136 are partly relaxed in conformation, which leads to disrupted active site microenvironments responsible for the activity loss and spectral change of NiRc-11. Comparison with the native structure revealed a bulky residue Met331 fastened by hydrogen bonding, which may play a direct role in keeping the right copper site geometry by protruding its side chain against the irregular coil 105-112. Sequence alignment showed that the bulky residue is conserved at position 331, indicating an equal importance of C-terminal segment in other copper-containing nitrite reductases.

Crystal structure of earthworm fibrinolytic enzyme component B: a novel, glycosylated two-chained trypsin.

The earthworm fibrinolytic enzyme (EFE), belonging to a group of serine proteases with strong fibrinolytic activity, has been used in a mixture as an oral drug for prevention and treatment of thrombosis in East Asia. The EFE component b (EFE-b) is one of seven EFE components from Eisenia fetida, and among them it has nearly the highest fibrinolytic activity. Here, we report its crystal structure at a resolution of 2.06A. The structural analysis shows that EFE-b should be classified as a trypsin from earthworm. However, it is distinct from other trypsins. It is a two-chained protease with an N-terminal, pyroglutamated light chain and an N-glycosylated heavy chain. Furthermore, the heavy chain contains a novel structural motif, an eight-membered ring resulting from a disulfide bridge between two neighboring cysteine residues, and a cis peptide bond exists between these two cysteine residues. The crystal structure of EFE-b provides the structural basis for its high level of stability and reveals its complicated post-translational modifications in earthworm. This structure is the first reported for a glycosylated two-chained trypsin, which may provide useful clues to explain the origin and evolution of the chymotrypsin family.

Structural basis for broad substrate specificity of earthworm fibrinolytic enzyme component A.

Earthworm fibrinolytic enzyme component A (EFE-a) possesses an S1 pocket, which is typical for an elastase-like enzyme, but it can still hydrolyze varieties of substrates, and it exhibits wide substrate specificity. Former structure studies suggested that the four-residue insertion after Val(217) might endow EFE-a with this specificity. Based on the native crystal structure at a resolution of 2.3A, we improved the native crystal structure to 1.8A and determined its complex structure with the inhibitor Meo-Suc-Ala-Ala-Pro-Val-CMK at a resolution of 1.9A. The final structures show that: (1) EFE-a possesses multisubstrate-binding sites interacting with the substrates; (2) significant conformation adjustment takes place at two loops binding to the N-terminal of the substrates, which may enhance the interaction between the enzyme and the substrates. These characteristics make the substrate-specificity of EFE-a less dependent on the property of its S1-pocket and may endow the enzyme with the ability to hydrolyze chymotrypsin-specific substrates and even trypsin-specific substrates.

Crystallization and preliminary crystallographic analysis of earthworm fibrinolytic enzyme component B from Eisenia fetida.

Earthworm fibrinolytic enzyme component B (EFE-b) is one of three high-fibrinolytic components found in Eisenia fetida. Several crystal forms were obtained by the hanging-drop vapour-diffusion technique. Diffraction data were collected from two well diffracting crystal forms. Crystal form I diffracted to a resolution of 2.25 A but was merohedrally twinned, with space group P2(1)3, unit-cell parameters a = b = c = 122.9 A and two molecules per asymmetric unit. Crystal form II diffracted to beyond 2.06 A resolution and belonged to space group P6(3)22, with unit-cell parameters a = b = 96.4, c = 150.8 A and one molecule per asymmetric unit. Crystal form II was a true single crystal and is suitable for structure determination.

pH-profile crystal structure studies of C-terminal despentapeptide nitrite reductase from Achromobacter cycloclastes.

Crystal structures of C-terminal despentapeptide nitrite reductase (NiRc-5) from Achromobacter cycloclastes were determined from 1.9 to 2.3A at pH 5.0, 5.4, and 6.2. NiRc-5, that has lost about 30% activity, is found to possess quite similar trimeric structures as the native enzyme. Electron density and copper content measurements indicate that the activity loss is not caused by the release of type 2 copper (T2Cu). pH-profile structural comparisons with native enzyme reveal that the T2Cu active center in NiRc-5 is perturbed, accounting for the partial loss of enzyme activity. This perturbation likely results from the less constrained conformations of two catalytic residues, Asp98 and His255. Hydrogen bonding analysis shows that the deletion of five residues causes a loss of more than half the intersubunit hydrogen bonds mediated by C-terminal tail. This study shows that the C-terminal tail plays an important role in controlling the conformations around the T2Cu site at the subunit interface, and helps keep the optimum microenvironment of active center for the full enzyme activity of AcNiR.

Crystal structure of spinach major light-harvesting complex at 2.72 A resolution.

The major light-harvesting complex of photosystem II (LHC-II) serves as the principal solar energy collector in the photosynthesis of green plants and presumably also functions in photoprotection under high-light conditions. Here we report the first X-ray structure of LHC-II in icosahedral proteoliposome assembly at atomic detail. One asymmetric unit of a large R32 unit cell contains ten LHC-II monomers. The 14 chlorophylls (Chl) in each monomer can be unambiguously distinguished as eight Chla and six Chlb molecules. Assignment of the orientation of the transition dipole moment of each chlorophyll has been achieved. All Chlb are located around the interface between adjacent monomers, and together with Chla they are the basis for efficient light harvesting. Four carotenoid-binding sites per monomer have been observed. The xanthophyll-cycle carotenoid at the monomer-monomer interface may be involved in the non-radiative dissipation of excessive energy, one of the photoprotective strategies that have evolved in plants.

Purification, characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida.

Seven fibrinolytic enzymes were purified from the earthworm Eisenia fetida. The molecular weights of the enzymes were 24663, 29516, 29690, 24201, 24170, 23028 and 29595, and the respective isoelectric points were 3.46, 3.5, 3.5, 3.68, 3.62, 3.94 and 3.46. All the proteases showed different fibrinolytic activity on fibrin plates. Studies on substrate specificity and inhibition indicated that they belonged to different types of serine proteases. N-Terminal sequencing indicated their high homology to those from the earthworm Lumbricus rubellus. All the enzymes have been crystallized.

Crystal structure studies on rubrerythrin: enzymatic activity in relation to the zinc movement.

Rubrerythrin (Rr) is a non-heme iron protein isolated from anaerobic sulfate-reducing bacteria. Rr is a dimeric molecule, each monomer contains a Fe(SCys)(4) center in the C-terminal domain and a binuclear metal center in the N-terminal domain. Rr structures with different protein sources and/or preparation procedures have been studied. Two Rr crystal structures have been solved with significant differences in their binuclear metal centers. The first structure, which was obtained from expressed protein under aerobic conditions, has a diiron-oxo center. The second structure, which was obtained from native protein of Desulfovibrio vulgaris under aerobic conditions, has an Fe-Zn center with the zinc position differing from the corresponding iron position in the former structure by approximately 2 A. The crystal structures of Rr isolated from D. vulgaris (Hildenborough, NCIB 8303), the same as the second structured but prepared under anaerobic conditions, are reported in this paper. The binuclear metal center in these structures is an Fe-Zn center. When the crystal was exposed to air, the zinc atom moved gradually, approximately 2 A, accompanied by the entrance of a water molecule (or hydroxyl group) and changes in the binuclear metal center microenvironment. This finding can explain the differences between the two different structures. The results suggest that the zinc movement may be related to the enzymatic activity of Rr.

Crystal structure of earthworm fibrinolytic enzyme component a: revealing the structural determinants of its dual fibrinolytic activity.

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida is a strong fibrinolytic enzyme that not only directly degrades fibrin, but also activates plasminogen. Proteolytic assays further revealed that it cleaved behind various P1 residue types. The crystal structure of EFEa was determined using the MIR method and refined to 2.3A resolution. The enzyme, showing the overall polypeptide fold of chymotrypsin-like serine proteases, possesses essential S1 specificity determinants characteristic of elastase. However, the beta strand at the west rim of the S1 specificity pocket is significantly elongated by a unique four-residue insertion (Ser-Ser-Gly-Leu) after Val217, which not only provides additional substrate hydrogen binding sites for distal P residues, but also causes extension of the S1 pocket at the south rim. The S2 subsite of the enzyme was partially occluded by the bulky side-chain of residue Tyr99. Structure-based inhibitor modeling demonstrated that EFEa's S1 specificity pocket was preferable for elastase-specific small hydrophobic P1 residues, while its accommodation of long and/or bulky P1 residues was also feasible if enhanced binding of the substrate and induced fit of the S1 pocket were achieved. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. The presence of Tyr99 at the S2 subsite indicates a preference for P2-Gly, while an induced fit of Tyr99 was also suggested for accommodation of bigger P2 residues. This structure is the first reported for an earthworm fibrinolytic enzyme component and serine protease originating from annelid worms.

Crystallization and preliminary crystallographic analysis of manganese superoxide dismutase from Bacillus halodenitrificans.

Manganese superoxide dismutase (GP-MnSOD), a component of the so-called 'green protein' (green protein complex) from the facultative anaerobic halodenitrifier Bacillus halodenitrificans, has been crystallized using the hanging-drop vapor diffusion method. Crystals have unit-cell parameters a=b=93.4 A, c=65.0 A, and belong to the space group P4(3)2(1)2. Preliminary analysis indicates there is one monomer in each asymmetric unit. The structural information from this enzyme will enrich our knowledge on its high catalytic activity and its possible role in green protein complex.

The crystallization and Preliminary Crystallographic Analysis of the Monoclinic Crystal Form of Basic Phospholipase A(2) from the Venom of Agkistrodon halys Pallas.

The basic phospholipase A(2) from the venom of Agkistrodon halys Pallas exhibits strong hemolytic activity. The enzyme has been crystallized by vapour diffusion techniques. Diffraction data of the crystal have been collected up to 2.5 Aring; resolution using the synchrotron radiation-imaging plate-Weissenberg camera system. The crystal parameters were calculated with an auto-indexing program. The crystal belongs to C2 space group with unit cell dimensions of alpha=100.38 Aring;, b=54.37 Aring;, c=117.38 Aring; and beta=120.71 deg;. Each asymmetric unit probably contains four or five molecules.