Functional and Evolutionary Relationship between Arginine Biosynthesis and Prokaryotic Lysine Biosynthesis through α-Aminoadipate

ABSTRACT Our previous studies revealed that lysine is synthesized through α-aminoadipate in an extremely thermophilic bacterium, Thermus thermophilus HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from α-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned anargD homolog of T. thermophilus HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of theargD-like gene, now termed lysJ, inT. thermophilus HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The lysJ gene was cloned into a plasmid and overexpressed in Escherichia coli, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the ɛ-amino group ofN 2-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. WhenN 2-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the δ-amino group ofN 2-acetylornithine to 2-oxoglutarate 16 times more efficiently than whenN 2-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in T. thermophilus HB27 is functionally and evolutionarily related to arginine biosynthesis.

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Insights into the Evolution of Macrolactam Biosynthesis through Cloning and Comparative Analysis of the Biosynthetic Gene Cluster for a Novel Macrocyclic Lactam, ML-449

Applied and Environmental Microbiology ◽

10.1128/aem.00744-09 ◽

2009 ◽

Vol 76 (1) ◽

pp. 283-293 ◽

Cited By ~ 51

Author(s):

Hanne Jørgensen ◽

Kristin F. Degnes ◽

Alexander Dikiy ◽

Espen Fjærvik ◽

Geir Klinkenberg ◽

...

Keyword(s):

Gene Cluster ◽

Evolutionary Relationship ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Side Chain ◽

Small Ring ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

The Difference ◽

High Level

ABSTRACT A new compound, designated ML-449, structurally similar to the known 20-membered macrolactam BE-14106, was isolated from a marine sediment-derived Streptomyces sp. Cloning and sequencing of the 83-kb ML-449 biosynthetic gene cluster revealed its high level of similarity to the BE-14106 gene cluster. Comparison of the respective biosynthetic pathways indicated that the difference in the compounds' structures stems from the incorporation of one extra acetate unit during the synthesis of the acyl side chain. A phylogenetic analysis of the β-ketosynthase (KS) domains from polyketide synthases involved in the biosynthesis of macrolactams pointed to a common ancestry for the two clusters. Furthermore, the analysis demonstrated the formation of a macrolactam-specific subclade for the majority of the KS domains from several macrolactam-biosynthetic gene clusters, indicating a closer relationship between macrolactam clusters than with the macrolactone clusters included in the analysis. Some KS domains from the ML-449, BE-14106, and salinilactam gene clusters did, however, show a closer relationship with KS domains from the polyene macrolide clusters, suggesting potential acquisition rather than duplication of certain PKS genes. Comparison of the ML-449, BE-14106, vicenistatin, and salinilactam biosynthetic gene clusters indicated an evolutionary relationship between them and provided new insights into the processes governing the evolution of small-ring macrolactam biosynthesis.

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Involvement of the arginine repressor in lysine biosynthesis of Thermus thermophilus

Microbiology ◽

10.1099/mic.0.29222-0 ◽

2006 ◽

Vol 152 (12) ◽

pp. 3585-3594 ◽

Cited By ~ 4

Author(s):

Kei Fujiwara ◽

Taishi Tsubouchi ◽

Tomohisa Kuzuyama ◽

Makoto Nishiyama

Keyword(s):

Growth Inhibition ◽

Gene Knockout ◽

Thermus Thermophilus ◽

Biosynthetic Gene Cluster ◽

Lysine Biosynthesis ◽

Dependent Manner ◽

Arginine Biosynthesis ◽

Knockout Mutant ◽

Mobility Shift ◽

Mobility Shift Assay

Lysine biosynthesis of Thermus thermophilus proceeds in a similar way to arginine biosynthesis, and some lysine biosynthetic enzymes from T. thermophilus so far investigated have the potential to function in arginine biosynthesis. These observations suggest that arginine might regulate the expression of genes for lysine biosynthesis. To test this hypothesis, the argR gene encoding the regulator of arginine biosynthesis was cloned from T. thermophilus and its function in lysine biosynthesis was analysed. The addition of arginine to the culture medium inhibited the growth of an arginase gene knockout mutant of T. thermophilus, which presumably accumulates arginine inside the cells. Arginine-dependent growth inhibition was not alleviated by the addition of ornithine, which is a biosynthetic intermediate of arginine and serves as a peptidoglycan component of the cell wall in T. thermophilus. However, the growth inhibition was cancelled either by the simultaneous addition of lysine and ornithine or by a knockout of the argR gene, suggesting the involvement of argR in regulation of lysine biosynthesis in T. thermophilus. Electrophoretic mobility shift assay and DNase I footprinting revealed that the ArgR protein specifically binds to the promoter region of the major lysine biosynthetic gene cluster. Furthermore, an α-galactosidase reporter assay for this promoter indicated that arginine repressed the promoter in an argR-dependent manner. These results indicate that lysine biosynthesis is regulated by arginine in T. thermophilus.

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Anacyclamide D8P, a prenylated cyanobactin from a Sphaerospermopsis sp. cyanobacterium

10.26434/chemrxiv.5346739.v1 ◽

2017 ◽

Cited By ~ 1

Author(s):

Joana Martins ◽

Niina Leikoski ◽

Matti Wahlsten ◽

Joana Azevedo ◽

Jorge Antunes ◽

...

Keyword(s):

Gene Cluster ◽

Cyclic Peptides ◽

Biosynthetic Gene Cluster ◽

The Novel ◽

Tyrosine Residue ◽

Biosynthetic Gene ◽

Post Translational Modification ◽

Biological Assays ◽

Mass Spectrometric Data ◽

Spectrometric Data

Cyanobactins are a family of linear and cyclic peptides produced through the post-translational modification of short precursor peptides. Anacyclamides are macrocyclic cyanobactins with a highly diverse sequence that are common in the genus Anabaena. A mass spectrometry-based screening of potential cyanobactin producers led to the discovery of a new prenylated member of this family of compounds, anacyclamide D8P (1), from Sphaerospermopsis sp. LEGE 00249. The anacyclamide biosynthetic gene cluster (acy) encoding the novel macrocyclic prenylated cyanobactin, was sequenced. Heterologous expression of the acy gene cluster in Escherichia coli established the connection between genomic and mass spectrometric data. Unambiguous establishment of the type and site of prenylation required the full structural elucidation of 1 using Nuclear Magnetic Resonance (NMR), which demonstrated that a forward prenylation occurred on the tyrosine residue. Compound 1 was tested in pharmacologically or ecologically relevant biological assays and revealed moderate antimicrobial activity towards the fouling bacterium Halomonas aquamarina CECT 5000.

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