5D84

Staphyloferrin B precursor biosynthetic enzyme SbnA bound to PLP

Summary for 5D84

• Entry DOI: 10.2210/pdb5d84/pdb
• Related: 5D85 5D86 5D87
• Descriptor: Probable siderophore biosynthesis protein SbnA, PYRIDOXAL-5'-PHOSPHATE, MAGNESIUM ION, ... (4 entities in total)
• Functional Keywords: siderophore, iron, plp, biosynthetic protein
• Biological source: Staphylococcus aureus
• Total number of polymer chains: 1
• Total formula weight: 36211.61

Authors

Grigg, J.C.,Kobylarz, M.J.,Liu, Y.,Lee, M.S.F.,Heinrichs, D.E.,Murphy, M.E.P. (deposition date: 2015-08-15, release date: 2016-02-03, Last modification date: 2020-01-08)

Primary citation

Kobylarz, M.J.,Grigg, J.C.,Liu, Y.,Lee, M.S.,Heinrichs, D.E.,Murphy, M.E.
Deciphering the Substrate Specificity of SbnA, the Enzyme Catalyzing the First Step in Staphyloferrin B Biosynthesis.
Biochemistry, 55:927-939, 2016

PubMed 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.

PubMed: 26794841
DOI: 10.1021/acs.biochem.5b01045

Experimental method

X-RAY DIFFRACTION (1.45 Å)