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1. In Vitro Reconstitution of Fimsbactin Biosynthesis from Acinetobacter baumannii

   https://doi.org/10.1021/acschembio.2c00573  

   Yang, Jinping ; Wencewicz, Timothy A. ( July 2022 , ACS Chemical Biology)

   He, Chuan ; Seyedsayamdost, Mohammed R. (Ed.)

   Siderophores produced via non-ribosomal peptide synthetase (NRPS) pathways serve as critical virulence factors for many pathogenic bacteria. An improved knowledge of siderophore biosynthesis guides the development of inhibitors, vaccines, and other therapeutic strategies. Fimsbactin A is a mixed ligand siderophore derived from human pathogenic Acinetobacter baumannii that contains phenolate-oxazoline, catechol, and hydroxamate metal chelating groups branching from a central L-Ser tetrahedral unit via amide and ester linkages. Fimsbactin A is derived from two molecules of L-Ser, two molecules of 2,3-dihydroxybenzoic acid (DHB), and one molecule of L-Orn and is a product of the fbs biosynthetic operon. Here, we report the complete in vitro reconstitution of fimsbactin A biosynthesis in a cell-free system using purified enzymes. We demonstrate the conversion of L-Orn to N1-acetyl-N1-hydroxy-putrescine (ahPutr) via ordered action of FbsJ (decarboxylase), FbsI (flavin N-monooxygenase), and FbsK (N-acetyltransferase). We achieve conversion of L-Ser, DHB, and L-Orn to fimsbactin A using FbsIJK in combination with the NRPS modules FbsEFGH. We also demonstrate chemoenzymatic conversion of synthetic ahPutr to fimsbactin A using FbsEFGH and establish the substrate selectivity for the NRPS adenylation domains in FbsH (DHB) and FbsF (L-Ser). We assign a role for the type II thioesterase FbsM in producing the shunt metabolite 2-(2,3-dihydroxyphenyl)-4,5-dihydrooxazole-4-carboxylic acid (DHB-oxa) via cleavage of the corresponding thioester intermediate that is tethered to NRPS peptidyl carrier domains during biosynthetic assembly. We propose a mechanism for branching NRPS-derived peptides via amide and ester linkages via the dynamic equilibration of N-DHB-Ser and O-DHB-Ser thioester intermediates via hydrolysis of DHB-oxa thioester intermediates. We also propose a genetic signature for NRPS ‘branching’ in the presence of a terminating C-T-C motif (FbsG). 

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2. Kinetic and Structural Characterization of a Flavin-Dependent Putrescine N -Hydroxylase from Acinetobacter baumannii

   https://doi.org/10.1021/acs.biochem.2c00493  

   Lyons, Noah S. ; Bogner, Alexandra N. ; Tanner, John J. ; Sobrado, Pablo ( November 2022 , Biochemistry)
3. Total Synthesis of an All-1,2- cis -Linked Repeating Unit from the Acinetobacter baumannii D78 Capsular Polysaccharide

   https://doi.org/10.1021/acs.orglett.2c01034  

   Njeri, Dancan K. ; Ragains, Justin R. ( May 2022 , Organic Letters)
4. Fimsbactin and Acinetobactin Compete for the Periplasmic Siderophore Binding Protein BauB in Pathogenic Acinetobacter baumannii

   https://doi.org/10.1021/acschembio.8b01051  

   Bohac, Tabbetha J. ; Fang, Luting ; Giblin, Daryl E. ; Wencewicz, Timothy A. ( March 2019 , ACS Chemical Biology)
5. Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate

   https://doi.org/10.1021/acs.biochem.3c00640  

   De Vitto, Humberto ; Belfon, Kafi K. ; Sharma, Nandini ; Toay, Sarah ; Abendroth, Jan ; Dranow, David. M. ; Lukacs, Christine M. ; Choi, Ryan ; Udell, Hannah S. ; Willis, Sydney ; et al ( February 2024 , Biochemistry)

   Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 μM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin. 

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