Synthesis of L-2,3-Diaminopropionic Acid, a Siderophore and Antibiotic Precursor

Abstract

L-2,3-diaminopropionic acid (L-Dap) is an amino acid that is a precursor of antibiotics and staphyloferrin B a siderophore produced by Staphylococcus aureus. SbnA and SbnB are encoded by the staphyloferrin B biosynthetic gene cluster and are implicated in L-Dap biosynthesis. We demonstrate here that SbnA uses PLP and substrates O-phospho-L-serine and L-glutamate to produce a metabolite N-(1-amino-1-carboxyl-2-ethyl)-glutamic acid (ACEGA). SbnB is shown to use NAD(+) to oxidatively hydrolyze ACEGA to yield α-ketoglutarate and L-Dap. Also, we describe crystal structures of SbnB in complex with NADH and ACEGA as well as with NAD(+) and α-ketoglutarate to reveal the residues required for substrate binding, oxidation, and hydrolysis. SbnA and SbnB contribute to the iron sparing response of S. aureus that enables staphyloferrin B biosynthesis in the absence of an active tricarboxylic acid cycle.

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus

“…Expression of the SB Biosynthetic and Efflux Genes Requires SbnI-In accordance with the idea that SbnI affected synthesis of SB, despite having no clear enzymatic function in the biosynthetic pathway (20,21,27), and given that SbnI shares low sequence similarity to the DNA-binding proteins ParB and Spo0J, we theorized that SbnI may function as a regulatory protein that impacted expression of SB biosynthetic genes. To evaluate this, RNA levels were measured for each of the genes throughout the sbn operon, comparing levels in WT to those in H984.…”

“…Thus, the SbnI-dependent up-regulation of sbnD and downstream genes may serve to ensure expression of the efflux pump prior to complete synthesis of the SB molecule. The early steps of SB synthesis, which result in the formation of L-Dap by SbnA and SbnB (20,21), may also be important for SbnI function, and we are currently investigating the possibility that SB intermediates, including L-Dap, work as co-regulators by binding with SbnI.…”

“…Because WT S. aureus strains have the ability to produce both SA and SB under conditions of iron starvation, and because their activity can be readily detected in bioassay experiments (20,21,27,28), a disc diffusion bioassay was performed to examine SB production in RN6390 (WT), H984, and the complemented strain (H984 ϩ psbnI). For these experiments, bacteria were grown in cTMS medium which, as opposed to RPMI 1640 medium, does not result in inhibition of SA or SB.…”

“…SbnA and SbnB perform the first step in SB synthesis with the formation of the precursor molecules L-2,3-diaminopropionic acid (L-Dap) and ␣-ketoglutarate from O-phospho-L-serine and L-glutamate (21). SbnG acts as a citrate synthase to convert acetyl-CoA and oxaloacetate into citrate (27,28).…”

SbnG, a Citrate Synthase in Staphylococcus aureus

“…Although TCA cycle activity is essential for the synthesis of SA, SB is freed from this dependence by enzymes encoded in the sbn gene cluster, expressed under iron restriction and during glycolytic growth (15,16). Recently, SbnA and SbnB were shown to form a metabolic pathway that produces L-2,3-diaminopropionic acid and ␣-ketoglutarate from Ophospho-L-serine and L-glutamate (15).…”