Stanislav Kadlcik

Mutasynthesis of Lincomycin Derivatives with Activity against Drug-Resistant Staphylococci

ABSTRACT The lincomycin biosynthetic gene lmbX was deleted in Streptomyces lincolnensis ATCC 25466, and deletion of this gene led to abolition of lincomycin production. The results of complementation experiments proved the blockage in the biosynthesis of lincomycin precursor 4-propyl-l-proline. Feeding this mutant strain with precursor derivatives resulted in production of 4′-butyl-4′-depropyllincomycin and 4′-pentyl-4′-depropyllincomycin in high titers and without lincomycin contamination. Moreover, 4′-pentyl-4′-depropyllincomycin was found to be more active than lincomycin against clinical Staphylococcus isolates with genes determining low-level lincosamide resistance.

Adaptation of an L-Proline Adenylation Domain to Use 4- Propyl-L-Proline in the Evolution of Lincosamide Biosynthesis

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 accomodate 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.

Lincosamide Synthetase—A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism

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.

New Concept of the Biosynthesis of 4-Alkyl-L-Proline Precursors of Lincomycin, Hormaomycin, and Pyrrolobenzodiazepines: Could a γ-Glutamyltransferase Cleave the C–C Bond?

Structurally different and functionally diverse natural compounds – antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin – share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C–C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C–C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.

Diversity of Alkylproline Moieties in Pyrrolobenzodiazepines Arises from Postcondensation Modifications of a Unified Building Block

Anticancer pyrrolobenzodiazepines (PBDs) are one of several groups of natural products that contain unusual 4-alkyl-l-proline derivatives (APDs) in their structure. APD moieties of PBDs are characterized by high structural diversity achieved through unknown biosynthetic machinery. Based on LC-MS analysis of culture broths, feeding experiments, and protein assays, we show that APDs are not incorporated into PBDs in their final form as was previously hypothesized. Instead, a uniform building block, 4-propylidene-l-proline or 4-ethylidene-l-proline, enters the condensation reaction. The subsequent postcondensation steps are initiated by the introduction of an additional double bond catalyzed by a FAD-dependent oxidoreductase, which we demonstrated with Orf7 from anthramycin biosynthesis. The resulting double bond arrangement presumably represents a prerequisite for further modifications of the APD moieties. Our study gives general insight into the diversification of APD moieties of natural PBDs and provides proof-of-principle for precursor directed and combinatorial biosynthesis of new PBD-based antitumor compounds.

Evolution-guided adaptation of an adenylation domain substrate specificity to an unusual amino acid

Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R)-4-propyl-proline (PPL). Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains’ unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.

Novel pathway of 3-hydroxyanthranilic acid formation in limazepine biosynthesis reveals evolutionary relation between phenazines and pyrrolobenzodiazepines

Natural pyrrolobenzodiazepines (PBDs) form a large and structurally diverse group of antitumour microbial metabolites produced through complex pathways, which are encoded within biosynthetic gene clusters. We sequenced the gene cluster of limazepines and proposed their biosynthetic pathway based on comparison with five available gene clusters for the biosynthesis of other PBDs. Furthermore, we tested two recombinant proteins from limazepine biosynthesis, Lim5 and Lim6, with the expected substrates in vitro. The reactions monitored by LC-MS revealed that limazepine biosynthesis involves a new way of 3-hydroxyanthranilic acid formation, which we refer to as the chorismate/DHHA pathway and which represents an alternative to the kynurenine pathway employed for the formation of the same precursor in the biosynthesis of other PBDs. The chorismate/DHHA pathway is presumably also involved in the biosynthesis of PBD tilivalline, several natural products unrelated to PBDs, and its part is shared also with phenazine biosynthesis. The similarities between limazepine and phenazine biosynthesis indicate tight evolutionary links between these groups of compounds.

C-C bond cleavage in biosynthesis of 4-alkyl- L -proline precursors of lincomycin and anthramycin cannot precede C -methylation

Zhong et al.1 confirmed that γ-glutamyltranspeptidase (γ-GTs) homologs are capable of cleaving a C–C bond, which was previously inferred by Jiraskova et al.2 in 2016 in a study based on gene inactivation experiments. The intriguing C–C bond cleavage catalyzed by LmbA and Ant6 γ-GT homologs from the biosynthesis of lincomycin A and anthramycin, respectively, was conclusively documented by Zhong et al.1. However, assignment of 2/3 as the LmbA and Ant6 substrate and 4/5 as the reaction product is questionable for several reasons; most importantly, it contradicts the current state of knowledge of the biosynthesis of 4-alkyl-L-proline derivatives (ALDP or APD used in previous literature; Fig. 1a)2. Here, we argue that LmbA/Ant6 γ-GT homologs do not utilize 2/3, but intermediate 9/10, which was previously proposed to be the main native substrate of LmbA2 and which is biosynthesized from 2/3 by a C-methylation reaction. Consequently, the main LmbA/Ant6 product is not 4/5 but compound 12, which is a subject of isomerization in order to proceed towards the final ALDP of lincomycin A and anthramycin. Here, we bring evidence that 2/3 is not the main native substrate of LmbA/Ant6 γ-GT homologs, but of LmbW/Ant5 Cmethyltransferases. Indeed, we observed in vitro C-methylation of 2/3 by LmbW affording 9/10 and we also detected intermediate 9/10 in the cultivation broth of the ΔlmbA mutant of lincomycin producing strain Streptomyces lincolnensis (Fig. 1b). Even though the conversion of 2/3 into 9/10 by LmbW was only partial, it clearly showed that 2/3 serves as an LmbW/ Ant5 substrate. To support that conversion of 2/3 by LmbW is not a side reaction resulting from broader substrate specificity of LmbW and that its main native substrate is indeed 2/3 and not 4/ 5 as the work by Zhong et al.1 suggests, we carried out a bioinformatic analysis of LmbW/Ant5. We found out that LmbW/Ant5 and their homologs (SibZ3, HrmC4, and Por105) from the biosyntheses of other ALDPs are similar to ALDPunrelated C-methyltransferases MppJ with known structure6 and MrsA7 (26% identity to LmbW according to BLAST for both MppJ and MrsA along the whole sequence; sequence alignment of LmbW and MppJ is available in Supplementary Fig. 1). MppJ and MrsA methylate phenylpyruvic and 5-guanidino-2oxopentanoic acids, respectively, i.e., substrates structurally analogous to 2/3 and not 4/5. Furthermore, methylation of phenylpyruvic acid catalyzed by MppJ is part of the biosynthesis of β-methyl-L-phenylalanine from L-phenylalanine8. Instead of direct methylation of L-phenylalanine, the machinery requires to proceed via phenylpyruvic acid, indicating the importance of the α-keto(enol)-carboxylic moiety of phenylpyruvic acid for the MppJ-catalyzed methylation. We propose that the same applies also to LmbW/Ant5 because their substrate 2/3 also contains the α-keto(enol)-carboxylic moiety. Importantly, conversion of the analogous substrates of MppJ and LmbW/Ant5 through a common reaction mechanism is supported by comparison of the active sites of MppJ (based on the protein crystal structure)6 vs. LmbW (based on a homology model) depicted in Fig. 2. The α-keto(enol)-carboxylic moiety appears to play an important role in fixation of the substrate within the active site not only in the case of MppJ, but also LmbW/Ant5. All these enzymes share the residues important for α-keto(enol)-carboxylic moiety fixation as well as the methylation (four residues depicted in blue in Fig. 2c, d). In contrast to 9/10, intermediate 4/5 (proposed as the LmbA/Ant6 reaction product and thus the LmbW/Ant5 substrate by Zhong et al.1) does not possess the α-keto(enol)-carboxylic moiety for the substrate fixation in the active site. Moreover, the methylation of 4/5 would have to proceed through a different mechanism than reactions catalyzed by MppJ and MrsA, which would be inconsistent with the high conservation of the key catalytic residues within the active sites of MppJ and LmbW/Ant5. Based on the above-mentioned arguments, we claim that 2/3 is first C-methylated by LmbW/Ant5 and the reaction product 9/10 is utilized as a substrate of LmbA/ Ant6 γ-GT homologs. However, 2/3 can serve as a minor substrate of LmbA if the C-methylation step is omitted and lincomycin B9, a side product of lincomycin A biosynthesis, is formed. Similarly, 2/3 undergoes C–C bond cleavage if the C-methyltransferase is not encoded within the biosynthetic gene cluster, which applies to the biosynthesis of e.g., tomaymycin10,11 and DOI: 10.1038/s41467-018-05455-3 OPEN

Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes

In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence.