Escherichia coli ilvN Interacts with the FAD Binding Domain of ilvB and Activates the AHAS I Enzyme

The unique multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been exploited to generate polypeptide fragments which, when cloned and expressed, reassemble in the presence of cofactors to yield a catalytically competent enzyme. Multidimensional multinuclear NMR methods have been employed for obtaining near complete sequence specific NMR assignments for backbone HN, 15N, 13Cα and 13Cβ atoms of the FAD binding domain of ilvB on samples that were isotopically enriched in 2H, 13C and 15N. Unambiguous assignments were obtained for 169 of 177 backbone Cα atoms and 127 of 164 side chain Cβ atoms. The secondary structure determined on the basis of observed 13Cα secondary chemical shifts and sequential NOEs agrees well with the structure of this domain in the catalytic subunit of yeast AHAS. Binding of ilvN to the ilvBα and ilvBβ domains was studied by both circular dichroism and isotope edited solution nuclear magnetic resonance methods. Changes in CD spectra indicate that ilvN interacts with ilvBα and ilvBβ domains of the catalytic subunit and not with the ilvBγ domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvBβ and proximal to the intrasubunit ilvBα/ilvBβ domain interface. The implication of this interaction on the role of the regulatory subunit on the activity of the holoenzyme is discussed

Solution NMR studies of acetohydroxy acid synthase I: Identification of the sites of inter-subunit interactions using multidimensional NMR methods

The novel multidomain organization in the multimeric Escherichia coli AHAS I (ilvBN) enzyme has been dissected to generate polypeptide fragments. These fragments when cloned, expressed and purified reassemble in the presence of cofactors to yield a catalytically competent enzyme. Structural characterization of AHAS has been impeded due to the fact that the holoenzyme is prone to dissociation leading to heterogeneity in samples. Our approach has enabled the structural characterization using high-resolution nuclear magnetic resonance methods. Near complete sequence specific NMR assignments for backbone H-N, N-15, C-13 alpha and C-13(beta) atoms of the FAD binding domain of ilvB have been obtained on samples isotopically enriched in H-2, C-13 and N-15. The secondary structure determined on the basis of observed C-13(alpha) secondary chemical shifts and sequential NOEs indicates that the secondary structure of the FAD binding domain of E. coli AHAS large Subunit (ilvB) is similar to the structure of this domain in the catalytic subunit of yeast AHAS. Protein-protein interactions involving the regulatory subunit (ilvN) and the domains of the catalytic subunit (ilvB) were studied using circular dichroic and isotope edited solution nuclear magnetic resonance spectroscopic methods. Observed changes in circular dichroic spectra indicate that the regulatory subunit (ilvN) interacts with ilvB alpha and ilvB beta domains of the catalytic subunit and not with the ilvB gamma domain. NMR chemical shift mapping methods show that ilvN binds close to the FAD binding site in ilvB beta and proximal to the intrasubunit ilvB alpha/ilvB beta domain interface. The implication of this interaction on the role of the regulatory subunit oil the activity of the holoenzyme is discussed. NMR studies of the regulatory domains show that these domains are structured in solution. Preliminary evidence for the interaction of ilvN with the metabolic end product of the pathway, viz., valine is also presented

Cupriavidus gilardii Pneumonia in a Young Patient with Chronic Kidney Disease: A Case Report

Cupriavidus gilardii (C. gilardii) is a Gram-negative, motile, non sporulating, non lactose fermenting bacterium. It was first identified by Coenye et al., and has a complex taxonomy, often being misidentified as Wausteria gilardii, Ralstonia gilardii. It is commonly found in ecosystems containing plants and heavy metal-contaminated soil and is rarely isolated from clinical samples with no clear evidence of its clinical significance. The pathogenic nature of C. gilardii in respiratory ailments, particularly in patients with cystic fibrosis, is still unclear. This case report presents a 19-year-old female with Chronic Kidney Disease (CKD) who developed pneumonia caused by C. gilardii. The report also includes the sensitivity pattern of the bacterium to guide physicians in treating these rare pathogens

A Rare Case of Aerococcus viridans Meningitis in a Patient with Trigeminal Nerve Schwannoma

The genus Aerococcus spp. comprise microaerophilic, catalase-negative, Gram-positive cocci that show alpha-haemolytic growth on blood agar. They have a tendency to divide on two planes at a 90° angle, and rapid multiplication leads to the formation of Grampositive cocci in tetrads and irregular clusters. Aerococcus spp. are capable of causing invasive and fatal systemic illnesses, such as endocarditis, bactereamia, arthritis, and meningitis. Due to evolving diagnostic tools, it is now identified as a pathogen in a variety of disorders instead of being considered a contaminant. Most isolates are susceptible to penicillins, but there is increasing resistance to cephalosporins, ciprofloxacin, cotrimoxazole, clindamycin, vancomycin, and tetracycline. Here, authors present a rare case of Aerococcus viridans meningitis in a patient who underwent surgical excision of a left trigeminal Schwannoma, along with the drug susceptibility pattern resistant to most first-line antibiotics used against isolates from Streptococci spp., except doxycycline

Structural consequences of replacement of an α- helical pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein α -helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein α -helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 Å . In wild-type thioredoxin, P40 is located in the interior of helix two, a long a-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin

High level expression of peptides and proteins using cytochrome b5b_5 as a fusion host

A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome $b_5$ has been designed. The ability of cytochrome $b_5$ to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., $\alpha$ hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, $Na_V1.2$. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel puriWcation protocol has been designed to purify the fusion proteins using metal aYnity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome $b_5$ based fusion system for the production of peptides and small proteins (<15 kDa)

Structural consequences of replacement of an \alpha-helical Pro residue in Escherichia coli thioredoxin

While it is well known that introduction of Pro residues into the interior of protein $\alpha$-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein $\alpha$-helix. We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1–1.5 kcal/mol in the temperature range 280–330 K. This paper describes the structure of the P40S mutant at a resolution of 1.8 \AA. In wild-type thioredoxin, P40 is located in the interior of helix two, a long $\alpha$-helix that extends from residues 32 to 49 with a kink at residue 40. Structural differences between the wild-type and P40S are largely localized to the above helix. In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36. The helix remains kinked. In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39. However, these are absent in P40S. Instead, these main chain atoms are hydrogen bonded to water molecules. The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin

Role of Bacillus subtilis BacB in the Synthesis of Bacilysin*

Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (l-alanine-[2,3-epoxycyclohexano-4]-l-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of l-anticapsin. These data, however, were not consistent with the putative functional annotation for these proteins whereby BacA, a prephenate dehydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize l-anticapsin. Here we demonstrate that BacA is a decarboxylase that acts on prephenate. Further, based on the biochemical characterization and the crystal structure of BacB, we show that BacB is an oxidase that catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to l-anticapsin. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvic acid. A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis