In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of S. lincolnensis strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of ccbZ gene adjacent to ccbN, the counterpart of lmbN, suggesting the gene rearrangement, evident also from still active internal translation start in lmbN, and indicating the direction of lincosamide biosynthesis evolution. The in vitro test with LmbN-CP, LmbC and the newly identified S. lincolnensis phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a holo-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.
The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.
- MeSH
- Anti-Bacterial Agents biosynthesis MeSH
- Bacterial Proteins genetics metabolism MeSH
- Circular Dichroism MeSH
- Dihydroxyphenylalanine metabolism MeSH
- Escherichia coli enzymology genetics MeSH
- Gene Expression MeSH
- Heme chemistry metabolism MeSH
- Hemeproteins genetics metabolism MeSH
- Hydroxylation MeSH
- Lincomycin biosynthesis MeSH
- Multigene Family MeSH
- Recombinant Proteins genetics metabolism MeSH
- Streptomyces enzymology genetics MeSH
- Tyrosine 3-Monooxygenase genetics metabolism MeSH
- Tyrosine metabolism MeSH
- Chromatography, High Pressure Liquid MeSH
- Iron chemistry metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Chemical diversity: Two SAM-dependent N-methyltransferases-LmbJ from the biosynthesis of the antibiotic lincomycin and CcbJ from celesticetin biosynthesis-have been characterized and compared. Both tested enzymes form multimers and are able to utilize N-demethyllincomycin, the natural substrate of LmbJ, with comparable efficiency.
- MeSH
- Anti-Bacterial Agents biosynthesis chemistry MeSH
- Biocatalysis * MeSH
- Lincomycin biosynthesis chemistry MeSH
- Lincosamides biosynthesis chemistry MeSH
- Methyltransferases chemistry metabolism MeSH
- Molecular Conformation MeSH
- Substrate Specificity MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Clinically used lincosamide antibiotic lincomycin incorporates in its structure 4-propyl-L-proline (PPL), an unusual amino acid, while celesticetin, a less efficient related compound, makes use of proteinogenic L-proline. Biochemical characterization, as well as phylogenetic analysis and homology modelling combined with the molecular dynamics simulation were employed for complex comparative analysis of the orthologous protein pair LmbC and CcbC from the biosynthesis of lincomycin and celesticetin, respectively. The analysis proved the compared proteins to be the stand-alone adenylation domains strictly preferring their own natural substrate, PPL or L-proline. The LmbC substrate binding pocket is adapted to accommodate a rare PPL precursor. When compared with L-proline specific ones, several large amino acid residues were replaced by smaller ones opening a channel which allowed the alkyl side chain of PPL to be accommodated. One of the most important differences, that of the residue corresponding to V306 in CcbC changing to G308 in LmbC, was investigated in vitro and in silico. Moreover, the substrate binding pocket rearrangement also allowed LmbC to effectively adenylate 4-butyl-L-proline and 4-pentyl-L-proline, substrates with even longer alkyl side chains, producing more potent lincosamides. A shift of LmbC substrate specificity appears to be an integral part of biosynthetic pathway adaptation to the PPL acquisition. A set of genes presumably coding for the PPL biosynthesis is present in the lincomycin--but not in the celesticetin cluster; their homologs are found in biosynthetic clusters of some pyrrolobenzodiazepines (PBD) and hormaomycin. Whereas in the PBD and hormaomycin pathways the arising precursors are condensed to another amino acid moiety, the LmbC protein is the first functionally proved part of a unique condensation enzyme connecting PPL to the specialized amino sugar building unit.
- MeSH
- Bacterial Proteins chemistry MeSH
- Dipeptides chemistry MeSH
- Lincomycin biosynthesis chemistry MeSH
- Lincosamides biosynthesis chemistry MeSH
- Evolution, Molecular * MeSH
- Molecular Dynamics Simulation * MeSH
- Streptomyces enzymology MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The biosynthetic pathway of the clinically important antibiotic lincomycin is not known in details. The precise knowledge of the lincomycin biosynthesis is a prerequisite for generation of improved derivatives by means of combinatorial genetics. Methods allowing determination of the key intermediates are very important tools of the pathway investigation. Two new high-performance liquid chromatography methods with fluorescence detection for determination of lincomycin precursors in fermentation broth of Streptomyces lincolnensis and its lincomycin nonproducing mutants were developed. The first one enables simultaneous analysis of methylthiolincosamide (MTL) and N-demethyllincomycin (NDL), whereas the second one is suitable for 4-propyl-L-proline (PPL) assay. Both methods are based on the pre-column derivatization: MTL and NDL with 4-chloro-7-nitrobenzofurazan; PPL with o-phthaldialdehyde. The methods were validated with lower limit of quantification values of 2.50, 3.75, and 3.75 microg ml(-1) for MTL, NDL, and PPL, respectively. The inter- and intra-day accuracies and precisions were all within 12%. Stability of oxidized and derivatized analytes was investigated.
- MeSH
- Amides analysis MeSH
- Fermentation MeSH
- Financing, Organized MeSH
- Fluorescence MeSH
- Lincomycin analogs & derivatives biosynthesis MeSH
- Molecular Structure MeSH
- Proline analogs & derivatives analysis MeSH
- Reproducibility of Results MeSH
- Streptomyces metabolism MeSH
- Sulfhydryl Compounds analysis MeSH
- Chromatography, High Pressure Liquid methods MeSH
A cosmid bearing an insert of 38 217 bp covering the gene cluster and its flanking regions of type strain Streptomyces lincolnensis ATCC 25466 was sequenced. Two relatively extensive sequence changes and several hundred point mutations were identified if compared with the previously published sequence of the lincomycin (Lin) industrial strain S. lincolnensis 78-11. Analysis of the cluster-flanking regions revealed its localization within the genome of the ATCC 25466 strain. The cluster-bearing cosmid was integrated into the chromosome of Lin non-producing strains S. coelicolor CH 999 and S. coelicolor M 145. The modified strains heterologously produced Lin but the level dropped to approximately 1-3% of the production in the ATCC 25466 strain.
- MeSH
- Anti-Bacterial Agents biosynthesis chemistry MeSH
- Bacterial Proteins genetics metabolism MeSH
- Biotechnology MeSH
- Point Mutation MeSH
- Gene Library MeSH
- Cosmids MeSH
- Lincomycin biosynthesis chemistry MeSH
- Multigene Family MeSH
- Sequence Analysis, DNA MeSH
- Streptomyces coelicolor genetics metabolism MeSH
- Streptomyces genetics metabolism growth & development MeSH
