The complete genome sequence of the nitrile biocatalyst Rhodocccus rhodochrous ATCC BAA-870.

BACKGROUND: Rhodococci are industrially important soil-dwelling Gram-positive bacteria that are well known for both nitrile hydrolysis and oxidative metabolism of aromatics. Rhodococcus rhodochrous ATCC BAA-870 is capable of metabolising a wide range of aliphatic and aromatic nitriles and amides. The genome of the organism was sequenced and analysed in order to better understand this whole cell biocatalyst. RESULTS: The genome of R. rhodochrous ATCC BAA-870 is the first Rhodococcus genome fully sequenced using Nanopore sequencing. The circular genome contains 5.9 megabase pairs (Mbp) and includes a 0.53 Mbp linear plasmid, that together encode 7548 predicted protein sequences according to BASys annotation, and 5535 predicted protein sequences according to RAST annotation. The genome contains numerous oxidoreductases, 15 identified antibiotic and secondary metabolite gene clusters, several terpene and nonribosomal peptide synthetase clusters, as well as 6 putative clusters of unknown type. The 0.53 Mbp plasmid encodes 677 predicted genes and contains the nitrile converting gene cluster, including a nitrilase, a low molecular weight nitrile hydratase, and an enantioselective amidase. Although there are fewer biotechnologically relevant enzymes compared to those found in rhodococci with larger genomes, such as the well-known Rhodococcus jostii RHA1, the abundance of transporters in combination with the myriad of enzymes found in strain BAA-870 might make it more suitable for use in industrially relevant processes than other rhodococci. CONCLUSIONS: The sequence and comprehensive description of the R. rhodochrous ATCC BAA-870 genome will facilitate the additional exploitation of rhodococci for biotechnological applications, as well as enable further characterisation of this model organism. The genome encodes a wide range of enzymes, many with unknown substrate specificities supporting potential applications in biotechnology, including nitrilases, nitrile hydratase, monooxygenases, cytochrome P450s, reductases, proteases, lipases, and transaminases.

Conversion of sterically demanding α,α-disubstituted phenylacetonitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191.

The nitrilase from Pseudomonas fluorescens EBC191 converted 2-methyl-2-phenylpropionitrile, which contains a quaternary carbon atom in the α-position toward the nitrile group, and also similar sterically demanding substrates, such as 2-hydroxy-2-phenylpropionitrile (acetophenone cyanohydrin) or 2-acetyloxy-2-methylphenylacetonitrile. 2-Methyl-2-phenylpropionitrile was hydrolyzed to almost stoichiometric amounts of the corresponding acid. Acetophenone cyanohydrin was transformed to the corresponding acid (atrolactate) and amide (atrolactamide) at a ratio of about 3.4:1. The (R)-acid and the (S)-amide were formed preferentially from acetophenone cyanohydrin. A homology model of the nitrilase suggested that steric hindrance with amino acid residue Tyr54 could impair the binding or conversion of sterically demanding substrates. Therefore, several enzyme variants that carried mutations in the respective residues were generated and subsequently analyzed for the substrate specificity and enantioselectivity of the reactions. Enzyme variants that demonstrated increased relative activities for the conversion of acetophenone cyanohydrin were identified. The chiral analysis of these reactions demonstrated peculiar reaction kinetics, which suggested that the enzyme variants converted the nonpreferred (S)-enantiomer of acetophenone cyanohydrin with a higher reaction rate than that of the (preferred) (R)-enantiomer. Recombinant whole-cell catalysts that simultaneously produced the nitrilase from P. fluorescens EBC191 and a plant-derived (S)-oxynitrilase from cassava (Manihot esculenta) converted acetophenone plus cyanide at pH 4.5 to (S)-atrolactate and (S)-atrolactamide. These recombinant cells are promising catalysts for the synthesis of stable chiral quaternary carbon centers from ketones.

Three-dimensional reconstruction of Heterocapsa circularisquama RNA virus by electron cryo-microscopy.

Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major natural agent controlling the host population. The viral capsid is constructed from a single gene product. Electron cryo-microscopy revealed that the virus has a diameter of 34 nm and T = 3 symmetry. The 180 quasi-equivalent monomers have an unusual arrangement in that each monomer contributes to a 'bump' on the surface of the protein. Though the capsid protein probably has the classic 'jelly roll' ß-sandwich fold, this is a new packing arrangement and is distantly related to the other positive-sense ssRNA virus capsid proteins. The handedness of the structure has been determined by a novel method involving high resolution scanning electron microscopy of the negatively stained viruses and secondary electron detection.

Crystal structure of Type III glutamine synthetase: surprising reversal of the inter-ring interface.

Glutamine synthetases are ubiquitous, homo-oligomeric enzymes essential for nitrogen metabolism. Unlike types I and II, which are well described both structurally and functionally, the larger, type IIIs are poorly characterized despite their widespread occurrence. An understanding of the structural basis for this divergence and the implications for design of type-specific inhibitors has, therefore, been impossible. The first crystal structure of a GSIII enzyme, presented here, reveals a conservation of the GS catalytic fold but subtle differences in protein-ligand interactions suggest possible avenues for the design GSIII inhibitors. Despite these similarities, the divergence of the GSIII enzymes can be explained by differences in quaternary structure. Unexpectedly, the two hexameric rings of the GSIII dodecamer associate on the opposite surface relative to types I and II. The diversity of GS quaternary structures revealed here suggests a nonallosteric role for the evolution of the double-ringed architecture seen in all GS enzymes.

Arginine 15 stabilizes an S(N)Ar reaction transition state and the binding of anionic ligands at the active site of human glutathione transferase A1-1.

Arg15, conserved in class Alpha GSTs (glutathione transferases), is located at the interface between the G- and H-sites of the active site where its cationic guanidinium group might play a role in catalysis and ligand binding. Arg15 in human GSTA1-1 was replaced with a leucine and crystallographic, spectroscopic, thermodynamic and molecular docking methods were used to investigate the contribution made by Arg15 towards (i) the binding of glutathione (GSH) to the G-site, (ii) the pK(a) of the thiol group of GSH, (iii) the stabilization of an analog of the anionic transition state of the S(N)Ar reaction between 1-chloro-2,4-dinitrobenzene (CDNB) and GSH, and, (iv) the binding of the anionic non-substrate ligand 8-anilino-1-naphthalene sulphonate (ANS) to the H-site. While the R15L mutation substantially diminishes the CDNB-GSH conjugating activity of the enzyme, it has little effect on protein structure and stability. Arg15 does not contribute significantly towards the enzyme's affinity for GSH but does determine the reactivity of GSH by reducing the thiol's pK(a) from 7.6 to 6.6. The anionic sigma-complex formed between GSH and 1,3,5-trinitrobenzene is stabilized by Arg15, suggesting that it also stabilizes the transition state formed in the S(N)Ar reaction between GSH and CDNB. The trinitrocyclohexadienate moiety of the sigma-complex binds the H-site where the catalytic residue, Tyr9, was identified to hydrogen bond to an o-nitro group of the sigma-complex. The affinity for ANS at the H-site is decreased about 3-fold by the R15L mutation implicating the positive electrostatic potential of Arg15 in securing the organic anion at this site.

A new approach to possible substrate binding mechanisms for nitrile hydratase.

We combined normal mode analysis (NMA) with cavity calculations as a method to get more insight into static crystal structures. We used nitrile hydratase (NHase) as a case study, and the crystal structure of a complex of Pseudonocardia thermophila NHase (1UGP) with n-butyric acid was chosen as a reference structure. The reference structure was compared with the other available NHase crystal structures. Cavity calculations of the static structures showed the entrances to the active site and also a possible function of the N-terminal in the substrate selection of the Co-type NHase. When NMA was combined with cavity calculations, a closing-opening passage was observed. Analysis of low frequency modes combined with cavity calculations led us to propose "breathing" and "flip-flop" mechanisms which might be a key part of the substrate binding mechanism.

Tertiary interactions stabilise the C-terminal region of human glutathione transferase A1-1: a crystallographic and calorimetric study.

The C-terminal region in class Alpha glutathione transferase A1-1 (GSTA1-1), which forms an amphipathic alpha-helix (helix 9), is known to contribute to the catalytic and non-substrate ligand-binding functions of the enzyme. The region in the apo protein is proposed to be disordered which, upon ligand binding at the active-site, becomes structured and localised. Because Ile219 plays a pivotal role in the stability and localisation of the region, the role of tertiary interactions mediated by Ile219 in determining the conformation and dynamics of the C-terminal region were studied. Ligand-binding microcalorimetric and X-ray structural data were obtained to characterise ligand binding at the active-site and the associated localisation of the C-terminal region. In the crystal structure of the I219A hGSTA1-1.S-hexylglutathione complex, the C-terminal region of one chain is mobile and not observed (unresolved electron density), whereas the corresponding region of the other chain is localised and structured as a result of crystal packing interactions. In solution, the mutant C-terminal region of both chains in the complex is mobile and delocalised resulting in a hydrated, less hydrophobic active-site and a reduction in the affinity of the protein for S-hexylglutathione. Complete dehydration of the active-site, important for maintaining the highly reactive thiolate form of glutathione, requires the binding of ligands and the subsequent localisation of the C-terminal region. Thermodynamic data demonstrate that the mobile C-terminal region in apo hGSTA1-1 is structured and does not undergo ligand-induced folding. Its close proximity to the surface of the wild-type protein is indicated by the concurrence between the observed heat capacity change of complex formation and the type and amount of surface area that becomes buried at the ligand-protein interface when the C-terminal region in the apo protein assumes the same localised structure as that observed in the wild-type complex.

Fibrinogen is degraded and internalized during incubation with neutrophils, and fibrinogen products localize to electron lucent vesicles.

A biologically relevant relationship exists between neutrophils and coagulation processes. Several studies have focused on the ability of neutrophil proteases (both intracellular and membrane-associated) to degrade fibrinogen. The present study investigates the events following the interaction of activated neutrophils with soluble fibrinogen. During incubation of PMA-stimulated neutrophils with fibrinogen at 37 degrees C, fibrinogenolysis occurred, and degraded fibrinogen became associated with the neutrophil. Immunoelectron microscopy identified these fibrinogen products to be located within electron lucent vesicles, and not on the surface of the cell, suggesting that they are internalized. Although a specific interaction between fibrinogen and the neutrophil membrane might assist uptake, in the presence of physiological concentrations of fibrinogen, internalization occurred largely via a non-specific pinocytic process. Studies at low temperature revealed that both intact and degraded forms of fibrinogen can associate with neutrophils. The fibrinogen products detected intracellularly in experiments performed at 37 degrees C might represent uptake of degraded as well as intact forms of fibrinogen, the latter being rapidly degraded intracellularly. This route of fibrinogenolysis contributes minimally to the overall extent of the degradation process, the majority occurring extracellularly. Neutrophils thus possess a proteolytic mechanism for preventing accumulation of surface ligand, perhaps allowing them to evade the immunomodulatory effects of such ligands during inflammation.