Similarities in the genomic sequence and coat protein structure of plant virsuses

The genomic sequences of several RNA plant viruses including cucumber mosaic virus, brome mosaic virus, alfalfa mosaic virus and tobacco mosaic virus have become available recently. The former two viruses are icosahedral while the latter two are bullet and rod shaped, respectively in particle morphology. The non-structural 3a proteins of cucumber mosaic virus and brome mosaic virus have an amino acid sequence homology of 35% and hence are evolutionarily related. In contrast, the coat proteins exhibit little homology, although the circular dichroism spectrum of these viruses are similar. The non-coding regions of the genome also exhibit variable but extensive homology. Comparison of the brome mosaic virus and alfalfa mosaic virus sequences reveals that they are probably related although with a much larger evolutionary distance. The polypeptide folds of the coat protein of three biologically distinct isometric plant viruses, tomato Bushy stunt virus, southern bean mosaic virus and satellite tobacco necrosis virus have been shown to display a striking resemblance. All of them consist of a topologically similar 8-standard β-Barrel. The implications of these studies to the understanding of the evolution of plant viruses will be discussed.     

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4? resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coli CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes.     

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.     

Kinetic studies of bioactive products nitric oxide and 8-iso-PGF(2alpha) in Burkholderia pseudomallei infected human macrophages, and their role in the intracellular survival of these organisms

The oxidative response of Burkholderia pseudomallei and Escherichia coli infected macrophages from normal and melioidosis subjects was determined by measuring the production of nitric oxide which is one of the reactive nitrogen intermediates, and the activation state of these macrophages was determined by measuring the generation of 8-iso-PGF(2alpha), a bioactive product of free radical induced lipid peroxidation. Macrophages obtained from the melioidosis patients generated significantly lower levels of nitric oxide and 8-iso-PGF(2alpha) compared to macrophages obtained from the normal subjects (P<0.001). The reduced efficiency of the oxygen dependent microbicidal mechanism in macrophages of melioidosis patients may be one of the survival strategies developed by B. pseudomallei to remain viable intracellularly.     

Design and synthesis of potent RSK inhibitors

Utilizing the already described 3,4-bi-aryl pyridine series as a starting point, incorporation of a second ring system with a hydrogen bond donor and additional hydrophobic contacts yielded the azaindole series which exhibited potent, picomolar RSK2 inhibition and the most potent in vitro target modulation seen thus far for a RSK inhibitor. In the context of the more potent core, several changes at the phenol moiety were assessed to potentially find a tool molecule appropriate for in vivo evaluation.     

Characterization of recombinant diaminopropionate ammonia-lyase from Escherichia coli and Salmonella typhimurium

Diaminopropionate ammonia-lyase gene from Escherichia coli and Salmonella typhimurium was cloned and the overexpressed enzymes were purified to homogeneity. The k(cat) values, determined for the recombinant enzymes with DL-DAP, D-serine, and L-serine as substrates, showed that the enzyme from S. typhimurium was more active than that from E. coli and the K(m) values were found to be similar. The purified enzymes had an absorption maximum (lambda(max)) at 412 nm, typical of PLP dependent enzymes. A red shift in lambda(max) was observed immediately after the addition of 10mM DL-DAP, which returned to the original lambda(max) of 412 nm in about 4 min. This red shift might reflect the formation of an external aldimine and/or other transient intermediates of the reaction. The apoenzyme of E. coli and S. typhimurium prepared by treatment with L-cysteine could be partially (60%) reconstituted by the addition of PLP. The holo, apo, and the reconstituted enzymes were shown to be present as homo dimers by size exclusion chromatography.     

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation

Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.     

Identification of a discrete intermediate in the assembly/disassembly of physalis mottle tymovirus through mutational analysis.

Assembly intermediates of icosahedral viruses are usually transient and are difficult to identify. In the present investigation, site-specific and deletion mutants of the coat protein gene of physalis mottle tymovirus (PhMV) were used to delineate the role of specific amino acid residues in the assembly of the virus and to identify intermediates in this process. N-terminal 30, 34, 35 and 39 amino acid deletion and single C-terminal (N188) deletion mutant proteins of PhMV were expressed in Escherichia coli. Site-specific mutants H69A, C75A, W96A, D144N, D144N-T151A, K143E and N188A were also constructed and expressed. The mutant protein lacking 30 amino acid residues from the N terminus self-assembled to T=3 particles in vivo while deletions of 34, 35 and 39 amino acid residues resulted in the mutant proteins that were insoluble. Interestingly, the coat protein (pR PhCP) expressed using pRSET B vector with an additional 41 amino acid residues at the N terminus also assembled into T=3 particles that were more compact and had a smaller diameter. These results demonstrate that the amino-terminal segment is flexible and either the deletion or addition of amino acid residues at the N terminus does not affect T=3 capsid assembly. In contrast, the deletion of even a single residue from the C terminus (PhN188Delta1) resulted in capsids that were unstable. These capsids disassembled to a discrete intermediate with a sedimentation coefficent of 19.4 S. However, the replacement of C-terminal asparagine 188 by alanine led to the formation of stable capsids. The C75A and D144N mutant proteins also assembled into capsids that were as stable as the pR PhCP, suggesting that C75 and D144 are not crucial for the T=3 capsid assembly. pR PhW96A and pR PhD144N-T151A mutant proteins failed to form capsids and were present as heterogeneous aggregates. Interestingly, the pR PhK143E mutant protein behaved in a manner similar to the C-terminal deletion protein in forming unstable capsids. The intermediate with an s value of 19.4 S was the major assembly product of pR PhH69A mutant protein and could correspond to a 30mer. It is possible that the assembly or disassembly is arrested at a similar stage in pR PhN188Delta1, pR PhH69A and pR PhK143E mutant proteins.     

(5'S)-8,5'-cyclo-2'-deoxyguanosine is a strong block to replication, a potent pol V-dependent mutagenic lesion, and is inefficiently repaired in Escherichia coli

8,5'-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was <1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II(-) strain, whereas it increased considerably in a pol IV(-) strain. Remarkably, no progeny was recovered from a pol V(-) strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ~34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5'C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP > S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.     

Development of a multiplex PCR assay for rapid identification of Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex

We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.