Naslov
In vitro characterization of an FMN-oxidoreductase- dependent aminoacyl carrier protein hydroxylase AcdB

Autori
Simunović, Vesna ; Truman, Andrew ; Weygand- Đurašević, Ivana ; Gruić Sovulj, Ita

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
24. hrvatski skup kemičara i kemijskih inženjera, Mini-simpozij Vladimir Prelog : knjiga sažetaka = 24th Croatian Meeting of Chemists and Chemical Engineers, Mini-symposium Vladimir Prelog : book of abstracts / Ukić, Šime ; Bolanča, tomislav - Zagreb : Hrvatsko društvo kemijskih inženjera i tehnologa (HDKI), 2015, 157-158

ISBN
978-953-6894-54-3

Skup
24. hrvatski skup kemičara i kemijskih inženjera = 24th Croatian Meeting of Chemists and Chemical Engineers

Mjesto i datum
Zagreb, Hrvatska, 21.04.2015. - 24.04.2015

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
FMN-oxidoreductase-dependent aminoacyl carrier

Sažetak
We are interested in deciphering the biochemical function of a new family of bacterial paralogs of atypical seryl-tRNA synthetases from methanogenic Archea that transfer an amino acid to a carrier protein instead onto a transfer RNA (tRNA). Curiously, in addition to the switch in the type of the acceptor macromolecule, amino acid:carrier protein (aa:Cp) ligases display alteration and relaxation in the substrate specificity, such that amino acid:Cp ligase from Agrobacterium tumefaciens preferentially activates alanine, whereas serine and glycine are activated to a 100- fold lesser extent. In addition to a aa:Cp ligase and its cognate carrier protein, comprehensive bioinformatics analysis of aa:Cp ligase gene neighborhoods has revealedan additional highly conserved open reading frame (ORF) in a great number ofspecies thatlargely belong to the Alpha- and Beta-Proteobacteria. This ORF is invariably located between the genes coding for carrier protein and aa:Cp ligase and encodes for a protein with a conserved fold of acyl-CoA dehydrogenase that bind flavin cofactors, designated AcdB. However, initial experiments have failed to detect alanine dehydrogenase/oxidase activity. Cofactor binding studies using thermophoresis have indicated that AcdB shows dissociation constant (Kd) toward flavine mononucletide (FMN) in the high micromolar range whereas no binding affinity toward flavin adenine dinucleotide (FAD) was detected. Based on these results, we speculated that AcdB may be anaminoacyl, carrier proteinboundmonooxygenase/hydroxylase whose activity depends on the FMN-oxidoreductase for a supply of FMNH2 reducing equivalents. To test this premise, we have reconstitued the initial steps of the aa:Cp ligase pathway by using liquid chromatography coupled to the time of flight mass spectrophotometer (LC-TOF-MS).Changes in the molecular mass of the carrier protein upon the transfer of the amino acid from the aa:Cp ligase to the phoshopathenyl (Ppant) arm of the Cp were analyzed allowing us for the first time detection of Cp forms loaded with up to three amino acids (alanine, glycine or serine). To ascertain whether additional aminoacylation events take place on the Ppant arm of the Cp, or elsewhere on the protein, Cp forms carrying mono-, or di- aminoacyl substrates were subjected to MS-MS fragmentation, providing a firm evidence that theaminoacyl substrates were attached tothe Ppantarm of the Cp. Following the addition of purified AcdB and EasA, a well-characterized NADPH:FMN oxidoreductase from Aspergillus fumigatus involved in the biosynthesis of ergot alkaloids [2], several novel peaks displaying an increaseof 16 mass units compared to the mass of carrier protein loaded with one, two or three alanines were detected. Interestingly, in addition to hydroxylating alanine, AcdB also hydroxylates serine or glycine(s) attached to the Cp. Together, ourdata argue that: i) AcdB, like aa:Cp-ligase, has a relaxed substrate specificityii) AcdB hydroxylates only one of the aminoacids irrespective of the number ofaminoacylations per Cp iii)hydroxylation of alanine does not take place at C-3, leading to theformation of serine. Current experiments are underway that should clarify exactly which atomundergoes hydroxylation.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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