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Dirk Hoffmeister

Publications and source records attributed to Dirk Hoffmeister.

12 recordsLinked to original sources
PubMed2006

Genomic mining for Aspergillus natural products.

The genus Aspergillus is renowned for its ability to produce a myriad of bioactive secondary metabolites. Although the propensity of biosynthetic genes to form contiguous clusters greatly facilitates assignment of putative secondary metabolite genes in the completed Aspergillus genomes, such analysis cannot predict gene expression and, ultimately, product formation. To circumvent this deficiency, we have examined Aspergillus nidulans microarrays for expressed secondary metabolite gene clusters by using the transcriptional regulator LaeA. Deletion or overexpression of laeA clearly identified numerous secondary metabolite clusters. A gene deletion in one of the clusters eliminated the production of the antitumor compound terrequinone A, a metabolite not described, from A. nidulans. In this paper, we highlight that LaeA-based genome mining helps decipher the secondary metabolome of Aspergilli and provides an unparalleled view to assess secondary metabolism gene regulation.

Aspergillus nidulans↗
PubMed2005

Functional analyses of oxygenases in jadomycin biosynthesis and identification of JadH as a bifunctional oxygenase/dehydrase.

A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase alpha (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.

Amino Acid Sequence↗
PubMed2005

Generation of novel landomycins M and O through targeted gene disruption.

Two genes from Streptomyces cyanogenous S136 that encode the reductase LanZ4 and the hydroxylase LanZ5, which are involved in landomycin A biosynthesis, were characterized by targeted gene inactivation. Analyses of the corresponding mutants as well as complementation experiments have allowed us to show that LanZ4 and LanZ5 are responsible for the unique C-11-hydroxylation that occurs during landomycin biosynthesis. Compounds accumulated by the lanZ4/Z5 mutants are the previously described landomycin F and the new landomycins M and O.

Aminoglycosides↗
PubMed2004

Natural product glycorandomization.

glycorandomization is a chemoenzymatic strategy that overcomes the limitations in natural product derivatization associated with both solely chemistry-based approaches or in vivo engineering. In this article we present the basic strategies for glycorandomization development as a next-generation tool in drug discovery.

Carbohydrate Sequence↗
PubMed2003

Creation of the first anomeric D/L-sugar kinase by means of directed evolution.

Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as d-galacturonic acid, d-talose, l-altrose, and l-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.

Carbohydrate Sequence↗
PubMed2003

The C-Glycosyltransferase UrdGT2 is unselective toward d- and l-configured nucleotide-bound rhodinoses.

UrdGT2 is a d-olivosyltransferase from the metabolic pathway of urdamycin A, an angucycline antitumor and antimicrobial drug. The remarkable feature of this biocatalyst is its ability to set up C-glycosidic bonds. Using an in vivo system suitable to deliver the trideoxysugar rhodinose in both d- and l- configuration we could verify that both have been accepted as substrates and attached to the urdamycin polyketide backbone via a C-glycosidic bond. Regardless of the stereochemistry, these C-glycosides served as acceptor for a subsequent glycosylation step to yield the novel urdamycins R and S with di-rhodinosyl side chains at C-9 of the polyketide moiety.

Glycosyltransferases↗
PubMed2002

Engineered urdamycin glycosyltransferases are broadened and altered in substrate specificity.

Combinatorial biosynthesis is a promising technique used to provide modified natural products for drug development. To enzymatically bridge the gap between what is possible in aglycon biosynthesis and sugar derivatization, glycosyltransferases are the tools of choice. To overcome limitations set by their intrinsic specificities, we have genetically engineered the protein regions governing nucleotide sugar and acceptor substrate specificities of two urdamycin deoxysugar glycosyltransferases, UrdGT1b and UrdGT1c. Targeted amino acid exchanges reduced the number of amino acids potentially dictating substrate specificity to ten. Subsequently, a gene library was created such that only codons of these ten amino acids from both parental genes were independently combined. Library members displayed parental and/or a novel specificity, with the latter being responsible for the biosynthesis of urdamycin P that carries a branched saccharide side chain hitherto unknown for urdamycins.

Amino Acid Sequence↗