1. Volume 21, Issue 3, Pages 379-388 (March 2014)
Synthesis of L-2,3-Diaminopropionic Acid, a Siderophore and Antibiotic Precursor 
Marek J. Kobylarz, Jason C. Grigg, Shin-ichi J. Takayama, Dushyant K. Rai, David E. Heinrichs, Michael E.P. Murphy 
Chemistry & Biology 
Volume 21, Issue 3, Pages (March 2014)
DOI: /j.chembiol
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2. Chemistry & Biology 2014 21, 379-388DOI: (10. 1016/j. chembiol. 2013
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3. Figure 1 SbnA Condenses OPS and L-Glutamate to Generate ACEGA
(A) UV-visible absorption spectra of SbnA (black line), SbnA in complex with OPS (blue line) and after the addition of excess L-glutamate (red line). The spectra were recorded with 30 μM SbnA, 30 μM OPS, and 3 mM L-glutamate.
(B) SbnA catalyzes the increased release of inorganic phosphate from OPS in the presence of L-glutamate. After 2 min, 2 mM of various amino acids were added to the SbnA reaction mixture. All reactions were performed in triplicate.
(C) LC-ESI-MS analysis of the SbnA reaction for product formation. A mass ion species of 235 corresponded to the predicted mass of ACEGA was identified. All mass spectra were recorded with retention times between 4.6–5.5 min.
See also Figure S1.
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4. Figure 2 Proposed Biosynthetic Pathway for L-Dap by SbnA and SbnB
The structure of the intermediate N-(1-amino-1-carboxy-2-ethyl)-glutamic acid (ACEGA) was determined by NMR.
See also Figure S2 and Table S2.
Chemistry & Biology  , DOI: ( /j.chembiol )
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5. Figure 3 SbnB Degrades ACEGA to Generate L-Dap and α-KG
(A) SbnB activity was monitored via the generation of NADH. SbnB reactions were coupled with SbnA, OPS, and L-glutamate. After 2 min, a total of 50 nmol of NAD+ was added to each reaction mixture.
(B) The SbnB reaction was reversible in the presence of NADH, L-Dap, and α-KG. SbnB activity was measured by monitoring the consumption of NADH.
(C) α-KG detection by the 2,4-dinitrophenylhydrazine assay. Aliquots of the reaction mixtures were removed every minute and assayed for total ketone levels. Error bars indicate SD from the mean for three replicates.
(D) LC-ESI-MS analysis of the full SbnB reverse reaction for product formation and confirmation of the reaction intermediate. A mass ion species of 235 corresponded to the predicted mass of ACEGA was identified. The mass spectrum was recorded with a retention time between 7.3–8.9 min.
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6. Figure 4
L-Dap and α-KG Generated from SbnA and SbnB Can Be Used by Sbn Siderophore Synthetases to Create SB In Vitro
Disk diffusion growth assays were performed with TMS agar plates seeded with S. aureus. SB enzyme reaction mixtures were spotted onto sterile paper disks and the diameter of growth was measured 48 hr after incubation. The full reaction sample contains enzymes SbnA and SbnB and the Staphyloferrin B sample is the positive control containing α-KG and L-Dap. Error bars were recorded as the SD for the mean from three replicates. The dashed line represents the diameter of the paper disk. The experiment was repeated using a S. aureus sirA mutant as a negative control; SirA is the lone receptor for ferric bound SB. See also Figure S3.
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7. Figure 5 Structure of SbnB
(A) The overall fold of apo-SbnB with the backbone shown as a schematic diagram. Dimerization and NAD binding domains colored green and orange, respectively.
(B) The SbnB dimer as obtained through crystallographic symmetry. Individual SbnB monomers are colored blue and green.
(C) α-KG bound into the active site of SbnB in the presence of NAD+. Omit difference electron density is shown as a gray mesh contoured at 1.0 σ. All residues and ligands are shown as sticks and the dashed line represents the distance between the C2 atom of α-KG and C4 atom of NAD+.
(D) ACEGA bound into the active site of SbnB in the presence of NADH. Omit difference electron density is shown as a gray mesh contoured at 1.0 σ. All residues and ligands are shown as sticks and the dashed line represents the distance between the C2 atom of ACEGA and C4 atom of NADH.
See also Figure S4 and Table S2.
Chemistry & Biology  , DOI: ( /j.chembiol )
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8. Figure 6 Structural Overlay of Homologs to SbnB
Structural overlays were assembled for SbnB against two homologs (A) alanine dehydrogenase (tan) from Archaeoglobus fulgidus (Protein Data Bank [PDB] ID: 1OMO) and (B) ornithine cyclodeaminase (purple) from Pseudomonas putida (PDB ID: 1X7D). SbnB ligands and residues were colored green with the exception of NADH, which was colored white. All active site residues and ligands are shown as sticks.
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9. Figure 7
Proposed Catalytic Mechanism for Oxidative Hydrolysis of ACEGA to Generate L-Dap and α-KG in SbnB
The first step involves a hydride transfer from the C2 atom of the L-glutamate-derived portion of ACEGA to NAD+. A water molecule attacks the resulting imine at the C2 atom, facilitated by the base, B, to form a carbinolamine intermediate. Collapse of the carbinolamine intermediate results in the formation of α-KG and L-Dap. See also Figure S5.
Chemistry & Biology  , DOI: ( /j.chembiol )
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