Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa

Murugan, Elavazhagan; Kong, Rong; Sun, Huihua; Rao, Feng; Liang, Zhao‐Xun

doi:10.1016/j.pep.2010.01.007

Cited by 25 publications

(32 citation statements)

References 25 publications

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“…The P. aeruginosa PAO1 AcpH gene [genID: 881435] identified previously 17 was cloned as described previously. 13 All primers used for cloning are located in supplemental information (Supplementary Table 2) Cyanothece PCC 7822 AcpH gene [genID: 9739974], and Shewanella oneidensis AcpH gene [genID: 1170805] were cloned from genomic DNA using standard techniques.…”

Section: Methodsmentioning

confidence: 99%

“…5,11,12,16 Despite an otherwise unknown natural role for the AcpH, an enzyme not consistently present in all bacteria, the identification of a more stable AcpH homolog from P. aeruginosa 17 and its subsequent characterization using free and fusion-ACPs with phosphopantetheine analogs 13 offered the potential of a promiscuous AcpH with which to establish a robust reversible labeling strategy. However, while this PaAcpH has primarily demonstrated promiscuity for a broad range of modified phosphopantetheines appended to the E. coli type II FAS ACP, we were unable to constitute activity on many carrier proteins in our library.…”

Section: Introductionmentioning

confidence: 99%

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Chemoenzymatic exchange of phosphopantetheine on protein and peptide

2014

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Evaluation of new acyl carrier protein hydrolase (AcpH, EC 3.1.4.14) homologs from proteobacteria and cyanobacteria reveals significant variation in substrate selectivity and kinetic parameters for phosphopantetheine hydrolysis from carrier proteins. Evaluation with carrier proteins from both primary and secondary metabolic pathways reveals an overall preference for acyl carrier protein (ACP) substrates from type II fatty acid synthases, as well as variable activity for polyketide synthase ACPs and peptidyl carrier proteins (PCP) from non-ribosomal peptide synthases. We also demonstrate the kinetic parameters of these homologs for AcpP and the 11-mer peptide substrate YbbR. These findings enable the fully reversible labeling of all three classes of natural product synthase carrier proteins as well as full and minimal fusion protein constructs.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Chemoenzymatic exchange of phosphopantetheine on protein and peptide

2014

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“…[18] Recently, the Pseudomonas aeruginosa AcpH has been developed as a much more amenable alternative to generate apo -ACP. [48] Both resin-attached and free protein techniques have been developed, allowing for the preparation of large quantities of apo -ACP and the recycling of high-value, isotopically enriched ACPs for NMR use. [49,50] Reversible tagging has also allowed for quantitative “apo-fication” of holo - and crypto-ACPs, yielding homogenous apo -ACP for further modification (Figure 2b).…”

Section: The Acyl Carrier Protein – An Extended Toolkitmentioning

confidence: 99%

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

2015

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Fatty acid biosynthesis is essential to life and represents one of the most conserved pathways in Nature, preserving the same handful of chemical reactions over all species. Recent interest in the molecular details of the de novo fatty acid synthase (FAS) has been heightened by demand for renewable fuels and the emergence of multidrug resistant bacterial strains. Central to FAS is the acyl carrier protein (ACP), a protein chaperone that shuttles the growing acyl chain between catalytic enzymes within the FAS. Human efforts to alter fatty acid biosynthesis for oil production, chemical feedstock or antimicrobial purposes has been met with limited success in part due to a lack of detailed molecular information behind the ACP-partner protein interactions inherent to the pathway. This review will focus on recently developed tools for the modification of ACP and analysis of protein-protein interactions, such as mechanism-based crosslinking, and the studies exploiting them. Discussion specific to each enzymatic domain focuses first on mechanism and known inhibitors, followed by available structures and known interactions with ACP. While significant unknowns remain, new understandings into the intricacies of FAS point to future advances in manipulating this complex molecular factory.

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“…In addition, makA6 and makA7, which are located downstream of the maklamicin PKS genes makA1-makA3, encode 4 -phosphopantetheinyl transferase and phosphoesterase, respectively. These two genes may be required for turnover of the 4 -phosphopantetheinyl group during the activation process of ACP domains (Murugan et al 2010). Of the 11 predicted dehydratase (DH) domains found in the PKS modules, 2 DH domains (DH1 and DH2) contain a mutation in the conserved motif in which the active site His is replaced by other residues (Fig.…”

Section: Genes Involved In the Polyketide Assembly Of Maklamicinmentioning

confidence: 99%

Characterization of the biosynthetic gene cluster for maklamicin, a spirotetronate-class antibiotic of the endophytic Micromonospora sp. NBRC 110955

Daduang

Kitani

Hashimoto

et al. 2015

Microbiological Research

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Maklamicin, which is produced by the endophytic Micromonospora sp. NBRC 110955, is a spirotetronate-class antibiotic possessing anti-microbial activity against Gram-positive bacteria, and has several unique structural features different from other spirotetronates. Here we describe identification and characterization of the maklamicin biosynthetic (mak) gene cluster through draft genome sequencing, genomic library screening, and gene disruption. Sequence analysis revealed that a plausible maklamicin cluster resides in a 152 kb DNA region encoding 46 open reading frames, 24 of which can be assigned roles in the biosynthesis of polyketide backbone, spirotetronate or peripheral moieties, self-resistance and the regulation of maklamicin production. Disruption of the polyketide synthase (PKS) genes makA1 or makA4 resulted in a complete loss of maklamicin production, indicating that the type I modular PKS system is responsible for the biosynthesis of maklamicin. The mak gene cluster contained a set of biosynthetic genes for the formation of a tetronate moiety, which were found to be highly conserved in the gene clusters for spirotetronate antibiotics. Based on the estimated biosynthetic genes, we propose the biosynthetic pathway for maklamicin. Our findings provide not only insights on the biosynthetic mechanism of the unique structures in maklamicin, but also useful information to facilitate a comparative analysis of the spirotetronate biosynthetic pathways to expand the structural repertoire.

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Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa

Cited by 25 publications

References 25 publications

Chemoenzymatic exchange of phosphopantetheine on protein and peptide

Chemoenzymatic exchange of phosphopantetheine on protein and peptide

Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

Characterization of the biosynthetic gene cluster for maklamicin, a spirotetronate-class antibiotic of the endophytic Micromonospora sp. NBRC 110955

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