title: Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis.
abstract: Staphylococcus aureus assembles the siderophore, staphyloferrin B, from l-2,3-diaminopropionic acid (l-Dap), α-ketoglutarate, and citrate. Recently, SbnA and SbnB were shown to produce l-Dap and α-ketoglutarate from O-phospho-l-serine (OPS) and l-glutamate. SbnA is a pyridoxal 5'-phosphate (PLP)-dependent enzyme with homology to O-acetyl-l-serine sulfhydrylases; however, SbnA utilizes OPS instead of O-acetyl-l-serine (OAS), and l-glutamate serves as a nitrogen donor instead of a sulfide. In this work, we examined how SbnA dictates substrate specificity for OPS and l-glutamate using a combination of X-ray crystallography, enzyme kinetics, and site-directed mutagenesis. Analysis of SbnA crystals incubated with OPS revealed the structure of the PLP-α-aminoacrylate intermediate. Formation of the intermediate induced closure of the active site pocket by narrowing the channel leading to the active site and forming a second substrate binding pocket that likely binds l-glutamate. Three active site residues were identified: Arg132, Tyr152, Ser185 that were essential for OPS recognition and turnover. The Y152F/S185G SbnA double mutant was completely inactive, and its crystal structure revealed that the mutations induced a closed form of the enzyme in the absence of the α-aminoacrylate intermediate. Lastly, l-cysteine was shown to be a competitive inhibitor of SbnA by forming a nonproductive external aldimine with the PLP cofactor. These results suggest a regulatory link between siderophore and l-cysteine biosynthesis, revealing a potential mechanism to reduce iron uptake under oxidative stress.
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia , Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Marek J
authors.initials: MJ
authors.lastname: Kobylarz
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia , Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Grigg
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia , Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Yunan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia , Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Mathew S F
authors.initials: MS
authors.lastname: Lee
authors.forename: David E
authors.initials: DE
authors.lastname: Heinrichs
authors.affiliation: Department of Microbiology and Immunology, Life Sciences Institute, The University of British Columbia , Vancouver, British Columbia, Canada V6T 1Z3.
authors.forename: Michael E P
authors.initials: ME
authors.lastname: Murphy
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D002951:Citrates / Q000096:biosynthesis* / Q000737:chemistry; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D009952:Ornithine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D002951:Citrates; C083439:staphyloferrin B; C063851:staphyloferrin A; D009952:Ornithine