Biochemical characterization of a psychrophilic and halotolerant α–carbonic anhydrase from a deep-sea bacterium, <i>Photobacterium profundum</i>

Prokaryotic α–carbonic anhydrases (α-CA) are metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and proton. We had reported the first crystal structure of a pyschrohalophilic α–CA from a deep-sea bacterium, Photobacterium profundum SS9. In this manuscript, we report the first biochemical characterization of P. profundum α–CA (PprCA) which revealed several catalytic properties that are atypical for this class of CA's. Purified PprCA exhibited maximal catalytic activity at psychrophilic temperatures with substantial decrease in activity at mesophilic and thermophilic range. Similar to other α–CA's, Ppr9A showed peak activity at alkaline pH (pH 11), although, PprCA retained 88% of its activity even at acidic pH (pH 5). Exposing PprCA to varying concentrations of oxidizing and reducing agents revealed that N-terminal cysteine residues in PprCA may play a role in the structural stability of the enzyme. Although inefficient in CO2 hydration activity under mesophilic and thermophilic temperatures, PprCA exhibited salt-dependent thermotolerance and catalytic activity under extreme halophilic conditions. Similar to other well-characterized α–CA's, PprCA is also inhibited by monovalent anions even at low concentrations. Finally, we demonstrate that PprCA accelerates CO2 biomineralization to calcium carbonate under alkaline conditions

Protein Glycosylation in Helicobacter pylori: Beyond the Flagellins?

Glycosylation of flagellins by pseudaminic acid is required for virulence in Helicobacter pylori. We demonstrate that, in H. pylori, glycosylation extends to proteins other than flagellins and to sugars other than pseudaminic acid. Several candidate glycoproteins distinct from the flagellins were detected via ProQ-emerald staining and DIG- or biotin- hydrazide labeling of the soluble and outer membrane fractions of wild-type H. pylori, suggesting that protein glycosylation is not limited to the flagellins. DIG-hydrazide labeling of proteins from pseudaminic acid biosynthesis pathway mutants showed that the glycosylation of some glycoproteins is not dependent on the pseudaminic acid glycosylation pathway, indicating the existence of a novel glycosylation pathway. Fractions enriched in glycoprotein candidates by ion exchange chromatography were used to extract the sugars by acid hydrolysis. High performance anion exchange chromatography with pulsed amperometric detection revealed characteristic monosaccharide peaks in these extracts. The monosaccharides were then identified by LC-ESI-MS/MS. The spectra are consistent with sugars such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Ac) previously described on flagellins, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), bacillosamine derivatives and a potential legionaminic acid derivative (Leg5AmNMe7Ac) which were not previously identified in H. pylori. These data open the way to the study of the mechanism and role of protein glycosylation on protein function and virulence in H. pylori

Purification and Preliminary Crystallization of SSA_0908, a Putative Substrate-Binding Protein in \u3ci\u3eStreptococcus sanguinis\u3c/i\u3e

Poster presented at the 2023 SWOSU Research and Scholarly Activity fair. Streptococcus sanguinis is a pathobiont that is the leading cause of subacute infective endocarditis (SIE) in humans. Blood transit and attachment to cardiac vegetation is a prerequisite for SIE pathogenesis. While numerous studies have identified cell-surface adhesins in S. sanguinis, many suggested to be involved in SIE remain uncharacterized. One being SSA_0908, a putative ABC-transporter substrate binding proteins (SBP) with homology to CD0837, a SBP from Clostridiodes difficle implicated in host colonization and aromatic amino acid transport. Sequence analysis showed that residues involved in aromatic amino acid ligand binding is highly conserved in SSA_0908. Homology modeling of SSA_0908 revealed a type 1 periplasmic SBP fold with two a-b-a sandwich domains connected via a hinge-loop. The ligand binding pocket at the interface of the sandwich domains shows active site architecture similar to other aromatic amino acid SBP’s. Sequence and structural homology of SSA_0908 to other characterized aromatic amino acid transporters indicated that this protein may be involved in similar function in S. sanguinis. In order to further characterize SSA_0908, we have successfully over-expressed and purified this protein using affinity chromatography. Preliminary crystallization trials resulted in microcrystals in several conditions. We are currently optimizing crystallization conditions to grow diffraction quality crystals.https://dc.swosu.edu/rf_2023/1004/thumbnail.jp

SSA_0809 is a Homotrimeric, Reactive Intermediate Deaminase A (RidA) from an Opportunistic Pathogen, \u3ci\u3eStreptococcus sanguinis\u3c/i\u3e

Poster presented at the 2023 SWOSU Research and Scholarly Activity fair. Pictured here is student Adreana Aquino Reactive intermediate deaminase A (RidA) is a low-molecular weight protein in YjgF/YER057c/UK114 superfamily. The archetypal RidA subfamily is involved in amino acid metabolism and shown to catalyze the neutralization of toxic 2-amino acrylate (2AA) intermediates produced during amino acid catabolism. In Salmonella enterica, mutants lacking ridA exhibit physiological defects from the antagonistic interaction of 2AA with pyridoxal phosphate (PLP)-dependent enzymes. The importance of RidA and the incomplete understanding of metabolic networks affected by RidA led us to investigate its role in Streptococcus sanguinis, an opportunistic pathogen and the leading cause of subacute infective endocarditis in humans. BLAST analysis of S. sanguinis genome revealed a protein SSA_0809 with 50% identity to RidA from S. enterica. Biochemical studies on S. sanguinis SSA_0809, henceforth SsRidA, revealed its capacity of accelerating 2AA neutralization to pyruvate. To better understand SsRidA activity, the first crystal structure in a holoenzyme confirmation was solved at 1.97Å. The overall structure of SsRidA revealed a homotrimeric arrangement with active sites formed at the monomer interfaces, typical for this family. Active site electron density revealed the presence of ligand in only one active site leaving two active sites unoccupied. This incomplete ligand occupancy in SsRidA is still under investigation.https://dc.swosu.edu/rf_2023/1005/thumbnail.jp

RpoS-Regulated Genes of Escherichia coli Identified by Random lacZ Fusion Mutagenesis

RpoS is a conserved alternative sigma factor that regulates the expression of many stress response genes in Escherichia coli. The RpoS regulon is large but has not yet been completely characterized. In this study, we report the identification of over 100 RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild-type backgrounds. Forty-eight independent gene fusions were identified, including several in well-characterized RpoS-regulated genes, such as osmY, katE, and otsA. Many of the other fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS-regulated genes of unknown functions are likely important in nutrient scavenging

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

A High-Resolution Crystal Structure of a Psychrohalophilic α–Carbonic Anhydrase from <i>Photobacterium profundum</i> Reveals a Unique Dimer Interface

<div><p>Bacterial α–carbonic anhydrases (α-CA) are zinc containing metalloenzymes that catalyze the rapid interconversion of CO₂ to bicarbonate and a proton. We report the first crystal structure of a pyschrohalophilic α–CA from a deep-sea bacterium, <i>Photobacterium profundum</i>. Size exclusion chromatography of the purified <i>P</i>. <i>profundum</i> α–CA (PprCA) reveals that the protein is a heterogeneous mix of monomers and dimers. Furthermore, an “in-gel” carbonic anhydrase activity assay, also known as protonography, revealed two distinct bands corresponding to monomeric and dimeric forms of PprCA that are catalytically active. The crystal structure of PprCA was determined in its native form and reveals a highly conserved “knot-topology” that is characteristic of α–CA’s. Similar to other bacterial α–CA’s, PprCA also crystallized as a dimer. Furthermore, dimer interface analysis revealed the presence of a chloride ion (Cl^-) in the interface which is unique to PprCA and has not been observed in any other α–CA’s characterized so far. Molecular dynamics simulation and chloride ion occupancy analysis shows 100% occupancy for the Cl^- ion in the dimer interface. Zinc coordinating triple histidine residues, substrate binding hydrophobic patch residues, and the hydrophilic proton wire residues are highly conserved in PprCA and are identical to other well-studied α–CA’s.</p></div

Cartoon representation of the PprCA active site and oligomerization status of PprCA.

<p>(<b>A</b>) The highly conserved active site residues of PprCA showing Zinc ion in the center of the active site (gray sphere). The hydrophobic (Val117, Val127, Leu181, Val190 and Trp192), the hydrophilic (Tyr17, Asn69, Gln74, Thr182 and Thr183), and zinc coordinating residues (His96, His98 and His115) are similar to other well characterized α–CA’s. (<b>B</b>) Active site water network involved in proton transfer is shown as red spheres. 2Fo-Fc electron density contoured at 8.0σ (green) and 1.0σ in blue. (<b>C</b>) Purified PprCA was analyzed on a Hiprep 16/60 Sephacryl S-200 size-exclusion column that was equilibrated with 20 mM Tris pH 7.5 and 150 mM NaCl. Peaks A and B correspond to dimer and monomer, respectively. The elution fractions were analyzed by SDS-PAGE and stained with Coomassie Blue. A single ~35 kDa band was observed for both dimeric and monomeric PprCA. MW–Molecular-weight marker, lanes 43 to 51 (elution fractions representing PprCA dimer) and lanes 52 to 64 (elution fractions representing PprCA monomer). (<b>D</b>) Purified PprCA was separated under both reducing and non-reducing conditions on a SDS-PAGE. Lanes were loaded with PprCA from monomer fraction, dimer fraction and from a sample containing a mixture of monomers and dimers. The gels were stained either by Coomassie blue or subjected to protonography. Protonography was performed according to De Luca et al, 2015 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168022#pone.0168022.ref009" target="_blank">9</a>]. The gel was incubated in CO₂ enriched water for 5 to 15 seconds at room temperature. Appearance of distinctive yellow bands in gels subjected to protonography indicates both monomeric (~27 kDa) and dimeric (~58 kDa) PprCA is catalytically active.</p