Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

Lee, Jung-Kul; Ang, Ee Lui; Zhao, Huimin

doi:10.1128/jb.00259-06

Cited by 25 publications

(26 citation statements)

References 24 publications

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“…All three mutations identified in our study did not result in significant alteration in the side chain size or the hydrophobicity, and yet they significantly affected the binding of lauroyl-CoA. It is not atypical in protein engineering that small changes in active site residues play a significant role in altering protein substrate specificity (36,37). In the case of PhlD, more drastic changes of tunnel residues than what was found in this study might not even be desired because they could affect the correct folding of the tunnel and consequently damage the overall activity of PhlD.…”

Section: Discussionmentioning

confidence: 68%

Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase fromPseudomonas fluorescens

Zha

Rubin-Pitel

Zhao³

2006

J. Biol. Chem.

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PhlD, a type III polyketide synthase from Pseudomonas fluorescens, catalyzes the synthesis of phloroglucinol from three molecules of malonyl-CoA. Kinetic analysis by direct measurement of the appearance of the CoASH product (k cat ‫؍‬ 24 ؎ 4 min ؊1 and K m ‫؍‬ 13 ؎ 1 M) gave a k cat value more than an order of magnitude higher than that of any other known type III polyketide synthase. PhlD exhibits broad substrate specificity, accepting C 4 -C 12 aliphatic acyl-CoAs and phenylacetyl-CoA as the starters to form C6-polyoxoalkylated ␣-pyrones from sequential condensation with malonyl-CoA. Interestingly, when primed with long chain acyl-CoAs, PhlD catalyzed extra polyketide elongation to form up to heptaketide products. A homology structural model of PhlD showed the presence of a buried tunnel extending out from the active site to assist the binding of long chain acyl-CoAs. To probe the structural basis for the unusual ability of PhlD to accept long chain acyl-CoAs, both site-directed mutagenesis and saturation mutagenesis were carried out on key residues lining the tunnel. Three mutations, M21I, H24V, and L59M, were found to significantly reduce the reactivity of PhlD with lauroyl-CoA while still retaining its physiological activity to synthesize phloroglucinol. Our homology modeling and mutational studies indicated that even subtle changes in the tunnel volume could affect the ability of PhlD to accept long chain acyl-CoAs. This suggested novel strategies for combinatorial biosynthesis of unnatural pharmaceutically important polyketides.

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Section: Discussionmentioning

confidence: 68%

Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase fromPseudomonas fluorescens

Zha

Rubin-Pitel

Zhao³

2006

J. Biol. Chem.

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“…12 value is in the same range as the activity of the other known N-oxygenase PrnD. 17 The comparatively low in vitro value is partly due to the lower temperature but for the most part a consequence of the special reaction conditions. 13…”

Section: Structure and Activity Determinationmentioning

confidence: 91%

Structure and Action of the N-oxygenase AurF from Streptomyces thioluteus

Zocher¹,

Winkler²,

Hertweck³

et al. 2007

Journal of Molecular Biology

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“…PCRbased detection revealed that none of the 18 endophytic isolates harbored specific genes involved in the biosynthesis of the antibiotics 2,4-diacetylphloroglucinol, phenazines, and pyoluteorin; the lack of production of these three antibiotics was confirmed by RP-HPLC analysis of culture extracts of the 18 endophytic isolates (data not shown). PCR-based detection of prnD, one of the four genes involved in pyrrolnitrin biosynthesis (48), and TLC analyses revealed that the endophyte Pseudomonas sp. strain ESR94 and eight of the endophytic Burkholderia isolates produced the antibiotic pyrrolnitrin (Table 3; Fig.…”

Section: Resultsmentioning

confidence: 99%

Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates

Mendes

Pizzirani-Kleiner

Araújo

et al. 2007

Appl Environ Microbiol

218

114

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Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37°C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria.Brazil is one of the world's largest sugarcane producers and has considerable influence over the international sugar market. Brazilian sugarcane is primarily used to produce sugar and alcohol. Production has increased over time to approximately 26 million tons of sugar and 16 million m 3 of alcohol in 2006. Although ethanol has been used as an alternative source of fuel in Brazil since 1980, it is currently receiving worldwide interest as a biofuel to replace, at least in part, gasoline, thereby contributing to a reduction in carbon emissions (33). Consequently, sustaining and enhancing the growth and yield of sugarcane have become a major focus of research. The growth and performance of sugarcane in the field are adversely affected by a number of abiotic and biotic factors, including a wide range of fungal and bacterial diseases. Pokkah boeng, caused by the fungus Fusarium moniliforme, is one of the most widespread diseases and may cause serious yield losses in commercial sugarcane plantings (68). F. moniliforme can be disseminated horizontally by airborne spores or crop debris and vertically through seed pieces. Current control strategies involve the use of resistant varieties and fungicide applications. The efficacy of both control measures, however, is limited, and there is an increasing need for novel and environmentally sound strategies to control this and other diseases of sugarcane.The overall goal of this study was to isolate and characterize beneficial bacteria that are intimately associated with sugarcane and have the potential to control pathogens and to promote the growth and yie...

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Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis

Cited by 25 publications

References 24 publications

Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase fromPseudomonas fluorescens

Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase fromPseudomonas fluorescens

Structure and Action of the N-oxygenase AurF from Streptomyces thioluteus

Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates

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