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1.  Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity 
The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes.
The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-­aminotransferases.
PMCID: PMC3606037  PMID: 23519665
aminotransferases; transaminases; substrate specificity; industrial biocatalysis
2.  JLigand: a graphical tool for the CCP4 template-restraint library 
The CCP4 template-restraint library defines restraints for biopolymers, their modifications and ligands that are used in macromolecular structure refinement. JLigand is a graphical editor for generating descriptions of new ligands and covalent linkages.
Biological macromolecules are polymers and therefore the restraints for macromolecular refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are associated with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation. Structure-specific template restraints can be defined in a user’s additional restraint library. Here, JLigand, a new CCP4 graphical interface to LibCheck and REFMAC that has been developed to manage the user’s library and generate new monomer entries is described, as well as new entries for links and associated modifications.
PMCID: PMC3322602  PMID: 22505263
macromolecular refinement; restraint library; molecular graphics
3.  Bacillus licheniformis Anti-TRAP can assemble into two types of dodecameric particles with the same symmetry but inverted orientation of trimers 
Journal of Structural Biology  2010;170(1-2):127-133.
Anti-TRAP (AT) protein regulates expression of tryptophan biosynthetic genes by binding to the trp RNA-binding attenuation protein (TRAP) and preventing its interaction with RNA. Bacillus subtilis AT forms trimers that can either interact with TRAP or can further assemble into dodecameric particles. To determine which oligomeric forms are preserved in AT proteins of other Bacilli we studied Bacillus licheniformis AT which shares 66% sequence identity with the B. subtilis protein. We show that in solution B. licheniformis AT forms stable trimers. In crystals, depending on pH, such trimers assemble into two different types of dodecameric particles, both having 23 point group symmetry. The dodecamer formed at pH 6.0 has the same conformation as previously observed for B. subtilis AT. This dodecamer contains a large internal chamber with the volume of ∼700 Å3, which is lined by the side chains of twelve valine residues. The presence of the hydrophobic chamber hints at the possibility that the dodecamer formation could be induced by binding of a ligand. Interestingly, in the dodecamer formed at pH 8.0 all trimers are turned inside out relatively to the form observed at pH 6.0.
PMCID: PMC2896485  PMID: 20138150
X-ray crystallography; Transcription attenuation; Tryptophan biosynthesis; trp RNA-binding attenuation protein; Anti-TRAP protein; Oligomeric assembly
4.  Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein 
Defects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown.
We determined the crystal structure of the SBDS orthologue from Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner.
It is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function.
PMCID: PMC2695463  PMID: 19454024
5.  NCS-constrained exhaustive search using oligomeric models 
Three difficult MR cases are reported in which the orientation of a search oligomer or its internal parameters were determined and the oligomer was positioned according to the maximal value of the correlation coefficient in a series of translation searches. Such an exhaustive search was feasible because of constraints on the model parameters derived from the self-rotation function.
The efficiency of the cross-rotation function step of molecular replacement (MR) is intrinsically limited as it uses only a fraction of the Patterson vectors. Along with general techniques extending the boundaries of the method, there are approaches that utilize specific features of a given structure. In special cases, where the directions of noncrystallographic symmetry axes can be unambiguously derived from the self-rotation function and the structure of the homologue protein is available in a related oligomeric state, the cross-rotation function step of MR can be omitted. In such cases, a small number of yet unknown parameters defining the orientation of the oligomer and/or its internal organization can be optimized using an exhaustive search. Three difficult MR cases are reported in which these parameters were determined and the oligomer was positioned according to the maximal value of the correlation coefficient in a series of translation searches.
PMCID: PMC2394828  PMID: 18094472
molecular replacement; exhaustive search
6.  Papillomavirus E1 helicase assembly maintains an asymmetric state in the absence of DNA and nucleotide cofactors 
Nucleic Acids Research  2007;35(19):6451-6457.
Concerted, stochastic and sequential mechanisms of action have been proposed for different hexameric AAA+ molecular motors. Here we report the crystal structure of the E1 helicase from bovine papillomavirus, where asymmetric assembly is for the first time observed in the absence of nucleotide cofactors and DNA. Surprisingly, the ATP-binding sites adopt specific conformations linked to positional changes in the DNA-binding hairpins, which follow a wave-like trajectory, as observed previously in the E1/DNA/ADP complex. The protein's assembly thus maintains such an asymmetric state in the absence of DNA and nucleotide cofactors, allowing consideration of the E1 helicase action as the propagation of a conformational wave around the protein ring. The data imply that the wave's propagation within the AAA+ domains is not necessarily coupled with a strictly sequential hydrolysis of ATP. Since a single ATP hydrolysis event would affect the whole hexamer, such events may simply serve to rectify the direction of the wave's motion.
PMCID: PMC2095799  PMID: 17881379
7.  Crystallization and preliminary X-ray diffraction analysis of ω-amino acid:pyruvate transaminase from Chromobacterium violaceum  
An ω-amino acid:pyruvate transaminase from C. violaceum has been purified and crystallized in two crystal forms. The structure has been solved using molecular replacement.
The enzyme ω-transaminase catalyses the conversion of chiral ω-amines to ketones. The recombinant enzyme from Chromobacterium violaceum has been purified to homogeneity. The enzyme was crystallized from PEG 4000 using the microbatch method. Data were collected to 1.7 Å resolution from a crystal belonging to the triclinic space group P1, with unit-cell parameters a = 58.9, b = 61.9, c = 63.9 Å, α = 71.9, β = 87.0, γ = 74.6°. Data were also collected to 1.95 Å from a second triclinic crystal form. The structure has been solved using the molecular-replacement method.
PMCID: PMC2330129  PMID: 17277454
ω-transaminase; pyridoxal 5′-phosphate

Results 1-7 (7)