Mixing Up the Pieces of the Desferrioxamine B Jigsaw Defines the Biosynthetic Sequence Catalyzed by DesD

Abstract

Late-stage assembly of the trimeric linear siderophore desferrioxamine B (DFOB) native to Streptomyces pilosus involves two DesD-catalyzed condensation reactions between one N-acetyl-N-hydroxy-1,5-diaminopentane (AHDP) unit and two N-succinyl-N-hydroxy-1,5-diaminopentane (SHDP) units. AHDP and SHDP are products of DesBC-catalyzed reactions of the native diamine substrate 1,5-diaminopentane (DP). The sequence of DesD-catalyzed DFOB biosynthesis was delineated by analyzing the distribution of DFOB analogues and dimeric precursors assembled by S. pilosus in medium containing 1,4-diamino-2(E)-butene (E-DBE). Seven unsaturated DFOB analogues were produced that were partially resolved by liquid chromatography (LC). Mass spectrometry (MS) measurements reported on the combination of E-DBE- and DP-derived substrates in each trimer (uDFOA1 series, 1:2; uDFOA2 series, 2:1; uDFOA3, 3:0). MS/MS fragmentation patterns reported on the absolute position of the substrate derivative at the N-acetylated terminus, the internal region, or the amine terminus of the trimer. The uDFOA1 and uDFOA2 series each comprised three constitutional isomers (binary notation (DP-derived substrate "0," E-DBE-derived substrate "1"); direction, N-acetylated-internal-amine): uDFOA1[001], uDFOA1[010], uDFOA1[100]; and uDFOA2[011], uDFOA2[110], and uDFOA2[101]. E-DBE completely replaced DP in uDFOA3[111]. Relative concentrations of the uDFOA1 series were uDFOA1[001] ≫ uDFOA1[100] > uDFOA1[010] and of the uDFOA2 series, uDFOA2[101] > uDFOA2[011] ≫ uDFOA2[110]. Dimeric compounds assembled from one N-acetylated and one N-succinylated substrate derivative were detected as trimer precursors: dDFX[00-] ≫ udDFX[10-] > udDFX[01-] (d = dimer, vacant position "-"). Relative concentrations of all species were consistent with the biosynthetic sequence: (i) SHDP activation, (ii) condensation with AHDP to form AHDP-SHDP, (iii) SHDP activation, and (iv) condensation with AHDP-SHDP to form DFOB.

Dissecting the Mechanism of Oligomerization and Macrocyclization Reactions of NRPS‐Independent Siderophore Synthetases

“…This is in accordance with recent results from the Codd group, who established that the biosynthesis of endcapped desferrioxamine Bp roceeds via activation of the monomeric substrate HS [5]A and not via activation of the dimeric precursor pre[5+ +5]. [16] Since AvbD only produces [5+ +ab] but not [5+ +5+ +ab],i tc an be concluded that the Cs ite of AvbD in contrast to DesDi su nable to bind [5+ +ab].B ased on these results, we propose that the Cs ite of AvbD is more restrictivei n substrate acceptance than that of DesDa nd thus may only accept monomeric substrates. This difference between the C sites of DesD and AvbD may determine the number of oligomerization steps for each enzyme.…”

“…Thisi si nl ine with resultsf rom the Codd group who reportedi ncorporation of an unsaturated HS [4]A precursor into desferrioxamine B. [16] Since both AvbD and DesD accept non-natural substrates, we reasoned that this promiscuity for synthetic substrates may provideameans to dissect and investigate the mechanism of type CN IS synthetases in greater detail.…”

“…[15] It has also been reported that unsaturated precursors have been incorporated into dimerica nd trimerich ydroxamates by PubC and DesD, respectively. [16,25] We have now confirmed ab road spectrum multi-specificity for various synthetic substrates for DesD, which allowedt oe mploy ac hemical biology approach for dissecting and interrogating the mechanism of this enzyme class on the example of AvbD and DesD.…”

Azido‐Desferrioxamine Siderophores as Functional Click‐Chemistry Probes Generated in Culture upon Adding a Diazo‐Transfer Reagent

“…In the first step, DesA catalyzes the decarboxylation of l ‐lysine to produce 1,5‐diaminopentane (DP) for downstream processing by DesBCD. Subsequent steps involve mono‐ N ‐hydroxylation of DP to produce N ‐hydroxy‐DP (HDP; DesB), acetylation or succinylation of the N ‐hydroxy group of HDP to produce N ‐acetyl‐ N ‐hydroxy‐DP (AHDP) or N ‐succinyl‐ N ‐hydroxy‐DP (SHDP), respectively (DesC), and assembly of an AHDP‐SHDP heterodimer (DesD, round 1) with the condensation of a final SHDP unit to AHDP‐SHDP to form trimeric 1 (DesD, round 2) . The exogenous supply of diamine substrates to S. pilosus culture medium bypasses DesA and requires only functional DesBCD.…”

“…DP is the major native diamine substrate used in the biosynthesis of 1 and of other DFO‐type siderophores. 1,4‐Diaminobutane (DB), produced through the decarboxylation of l ‐ornithine, is a minor native diamine substrate used solely or in combination with DP to assemble DFOA‐type siderophores (Schemes and B) …”