ThnL, a B12-dependent radical S-adenosylmethionine enzyme, catalyzes thioether bond formation in carbapenem biosynthesis.

Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

The FloR master regulator controls flotation, virulence and antibiotic production in Serratia sp. ATCC 39006.

Serratia sp. ATCC 39006 produces intracellular gas vesicles to enable upward flotation in water columns. It also uses flagellar rotation to swim through liquid and swarm across semi-solid surfaces. Flotation and motility can be co-regulated with production of a ß-lactam antibiotic (carbapenem carboxylate) and a linear tripyrrole red antibiotic, prodigiosin. Production of gas vesicles, carbapenem and prodigiosin antibiotics, and motility are controlled by master transcriptional and post-transcriptional regulators, including the SmaI/SmaR-based quorum sensing system and the mRNA binding protein, RsmA. Recently, the ribose operon repressor, RbsR, was also defined as a pleiotropic regulator of flotation and virulence factor elaboration in this strain. Here, we report the discovery of a new global regulator (FloR; a DeoR family transcription factor) that modulates flotation through control of gas vesicle morphogenesis. The floR mutation is highly pleiotropic, down-regulating production of gas vesicles, carbapenem and prodigiosin antibiotics, and infection in Caenorhabditis elegans, but up-regulating flagellar motility. Detailed proteomic analysis using TMT peptide labelling and LC-MS/MS revealed that FloR is a physiological master regulator that operates through subordinate pleiotropic regulators including Rap, RpoS, RsmA, PigU, PstS and PigT.

Methylations in complex carbapenem biosynthesis are catalyzed by a single cobalamin-dependent radical S-adenosylmethionine enzyme.

Complex carbapenem ß-lactam antibiotics contain diverse C6 alkyl substituents constructed by cobalamin-dependent radical SAM enzymes. TokK installs the C6 isopropyl chain found in asparenomycin. Time-course analyses of TokK and its ortholog ThnK, which forms the C6 ethyl chain of thienamycin, indicate that catalysis occurs through a sequence of discrete, non-processive methyl transfers.

Metabolic engineering of a carbapenem antibiotic synthesis pathway in Escherichia coli.

Carbapenems, a family of ß-lactam antibiotics, are among the most powerful bactericidal compounds in clinical use. However, as rational engineering of native carbapenem-producing microbes is not currently possible, the present carbapenem supply relies upon total chemical synthesis of artificial carbapenem derivatives. To enable access to the full diversity of natural carbapenems, we have engineered production of a simple carbapenem antibiotic within Escherichia coli. By increasing concentrations of precursor metabolites and identifying a reducing cofactor of a bottleneck enzyme, we improved productivity by 60-fold over the minimal pathway and surpassed reported titers obtained from carbapenem-producing Streptomyces species. We stabilized E. coli metabolism against antibacterial effects of the carbapenem product by artificially inhibiting membrane synthesis, which further increased antibiotic productivity. As all known naturally occurring carbapenems are derived from a common intermediate, our engineered strain provides a platform for biosynthesis of tailored carbapenem derivatives in a genetically tractable and fast-growing species.

Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in ß-lactam biosynthesis.

Covering: up to 2017 2-Oxoglutarate (2OG) dependent oxygenases and the homologous oxidase isopenicillin N synthase (IPNS) play crucial roles in the biosynthesis of ß-lactam ring containing natural products. IPNS catalyses formation of the bicyclic penicillin nucleus from a tripeptide. 2OG oxygenases catalyse reactions that diversify the chemistry of ß-lactams formed by both IPNS and non-oxidative enzymes. Reactions catalysed by the 2OG oxygenases of ß-lactam biosynthesis not only involve their typical hydroxylation reactions, but also desaturation, epimerisation, rearrangement, and ring-forming reactions. Some of the enzymes involved in ß-lactam biosynthesis exhibit remarkable substrate and product selectivities. We review the roles of 2OG oxygenases and IPNS in ß-lactam biosynthesis, highlighting opportunities for application of knowledge of their roles, structures, and mechanisms.

Phylogenomic Analysis of ß-Lactamase in Archaea and Bacteria Enables the Identification of Putative New Members.

ß-lactamases are enzymes which are commonly produced by bacteria and which degrade the ß-lactam ring of ß-lactam antibiotics, namely penicillins, cephalosporins, carbapenems, and monobactams, and inactivate these antibiotics. We performed a rational and comprehensive investigation of ß-lactamases in different biological databases. In this study, we constructed hidden Markov model profiles as well as the ancestral sequence of four classes of ß-lactamases (A, B, C, and D), which were used to identify potential ß-lactamases from environmental metagenomic (1206), human microbiome metagenomic (6417), human microbiome reference genome (1310), and NCBI's nonredundant databases (44101). Our analysis revealed the existence of putative ß-lactamases in the metagenomic databases, which appeared to be similar to the four different molecular classes (A-D). This is the first report on the large-scale phylogenetic diversity of new members of ß-lactamases, and our results revealed that metagenomic database dark-matter contains ß-lactamase-like antibiotic resistance genes.

Mechanism of the C5 stereoinversion reaction in the biosynthesis of carbapenem antibiotics.

The bicyclic ß-lactam/2-pyrrolidine precursor to all carbapenem antibiotics is biosynthesized by attachment of a carboxymethylene unit to C5 of L-proline followed by ß-lactam ring closure. Carbapenem synthase (CarC), an Fe(II) and 2-(oxo)glutarate (Fe/2OG)-dependent oxygenase, then inverts the C5 configuration. Here we report the structure of CarC in complex with its substrate and biophysical dissection of its reaction to reveal the stereoinversion mechanism. An Fe(IV)-oxo intermediate abstracts the hydrogen (H•) from C5, and tyrosine 165, a residue not visualized in the published structures of CarC lacking bound substrate, donates H• to the opposite face of the resultant radical. The reaction oxidizes the Fe(II) cofactor to Fe(III), limiting wild-type CarC to one turnover, but substitution of the H•-donating tyrosine disables stereoinversion and confers to CarC the capacity for catalytic substrate oxidation.

Identification and characterization of the carbapenem MM 4550 and its gene cluster in Streptomyces argenteolus ATCC 11009.

Nearly 50 naturally occurring carbapenem ß-lactam antibiotics, most produced by Streptomyces, have been identified. The structural diversity of these compounds is limited to variance of the C-2 and C-6 side chains as well as the stereochemistry at C-5/C-6. These structural motifs are of interest both for their antibiotic effects and their biosynthesis. Although the thienamycin gene cluster is the only active gene cluster publically available in this group, more comparative information is needed to understand the genetic basis of these structural differences. We report here the identification of MM 4550, a member of the olivanic acids, as the major carbapenem produced by Streptomyces argenteolus ATCC 11009. Its gene cluster was also identified by degenerate PCR and targeted gene inactivation. Sequence analysis revealed that the genes encoding the biosynthesis of the bicyclic core and the C-6 and C-2 side chains are well conserved in the MM 4550 and thienamycin gene clusters. Three new genes, cmmSu, cmm17 and cmmPah were found in the new cluster, and their putative functions in the sulfonation and epimerization of MM 4550 are proposed. Gene inactivation showed that, in addition to cmmI, two new genes, cmm22 and -23, encode a two-component response system thought to regulate the production of MM 4550. Overexpression of cmmI, cmm22 and cmm23 promoted MM 4550 production in an engineered strain. Finally, the involvement and putative roles of all genes in the MM 4550 cluster are proposed based on the results of bioinformatics analysis, gene inactivation, and analysis of disruption mutants. Overall, the differences between the thienamycin and MM 4550 gene clusters are reflected in characteristic structural elements and provide new insights into the biosynthesis of the complex carbapenems.

Mechanistic insights into the bifunctional non-heme iron oxygenase carbapenem synthase by active site saturation mutagenesis.

The carbapenem class of ß-lactam antibiotics is known for its remarkable potency, antibacterial spectrum, and resistance to ß-lactamase-mediated inactivation. While the biosynthesis of structurally "complex" carbapenems, such as thienamycin, share initial biochemical steps with carbapenem-3-carboxylate ("simple" carbapenem), the requisite inversion at C5 and formation of the characteristic α,ß-unsaturated carboxylate are different in origin between the two groups. Here, we consider carbapenem synthase, a mechanistically distinct bifunctional non-heme iron α-ketoglutarate-dependent enzyme responsible for the terminal reactions, C5 epimerization and desaturation, in simple carbapenem production. Interestingly, this enzyme accepts two stereoisomeric substrates and transforms each to a common active antibiotic. Owing both to enzyme and product instability, resorting to saturation mutagenesis of active site and selected second-sphere residues gave clearly differing profiles of CarC tolerance to structural modification. Guided by a crystal structure and the mutational data, in silico docking was used to suggest the positioning of each disastereomeric substrate in the active site. The two orientations relative to the reactive iron-oxo center are manifest in the two distinct reactions, C5-epimerization and C2/3-desaturation. These observations favor a two-step reaction scheme involving two complete oxidative cycles as opposed to a single catalytic cycle in which an active site tyrosine, Tyr67, after hydrogen donation to achieve bicyclic ring inversion, is further hypothesized to serve as a radical carrier.

Crotonase catalysis enables flexible production of functionalized prolines and carbapenams.

The biocatalytic versatility of wildtype and engineered carboxymethylproline synthases (CMPSs) is demonstrated by the preparation of functionalized 5-carboxymethylproline derivatives methylated at C-2, C-3, C-4, or C-5 of the proline ring from appropriately substituted amino acid aldehydes and malonyl-coenzyme A. Notably, compounds with a quaternary center (at C-2 or C-5) were prepared in a stereoselective fashion by engineered CMPSs. The substituted-5-carboxymethyl-prolines were converted into the corresponding bicyclic ß-lactams using a carbapenam synthetase. The results demonstrate the utility of the crotonase superfamily enzymes for stereoselective biocatalysis, the amenability of carbapenem biosynthesis pathways to engineering for the production of new bicyclic ß-lactam derivatives, and the potential of engineered biocatalysts for the production of quaternary centers.

The stationary phase sigma factor, RpoS, regulates the production of a carbapenem antibiotic, a bioactive prodigiosin and virulence in the enterobacterial pathogen Serratia sp. ATCC 39006.

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and invertebrate animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, a prodigiosin and a carbapenem, and the exoenzymes pectate lyase and cellulase. We showed previously that deletion of the RNA chaperone Hfq abolished antibiotic production and attenuated virulence in both animal and plant hosts. Hfq and dependent small RNAs (sRNAs) are known to regulate the post-transcriptional expression of rpoS, which encodes σ(S), the stationary phase sigma factor subunit of RNA polymerase. An S39006 hfq deletion mutant showed decreased transcript levels of rpoS. Therefore, in this study we investigated whether the phenotypes regulated by Hfq were mediated through its control of rpoS. Whereas loss of Hfq abolished prodigiosin and carbapenem production and attenuated virulence in both C. elegans and potato, characterization of an S39006 rpoS mutant showed unexpectedly elevated prodigiosin and carbapenem production. Furthermore, the rpoS mutant exhibited attenuated animal pathogenesis, but not plant pathogenesis. Additionally, a homologue of the Hfq-dependent sRNA, RprA, was identified and shown to regulate prodigiosin production in a manner consistent with its role in positively regulating translation of rpoS mRNA. Combined, these results demonstrate that Hfq regulation of secondary metabolism and plant pathogenesis is independent of RpoS and establishes RpoS and RprA as regulators of antibiotic production.

Definition of the common and divergent steps in carbapenem ß-lactam antibiotic biosynthesis.

Approximately 50 naturally occurring carbapenem ß-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.

The RNA chaperone, Hfq, controls two luxR-type regulators and plays a key role in pathogenesis and production of antibiotics in Serratia sp. ATCC 39006.

Serratia sp. ATCC 39006 (S39006) is a Gram-negative bacterium that is virulent in plant (potato) and animal (Caenorhabditis elegans) models. It produces two secondary metabolite antibiotics, a prodigiosin and a carbapenem, and the exoenzymes, pectate lyase and cellulase. A complex regulatory network that includes quorum sensing (QS) controls production of prodigiosin. While many aspects of the regulation of the metabolites and exoenzymes are well understood, the potential role in this network of the RNA chaperone Hfq and dependent small regulatory RNAs has not been characterized. Hfq is an RNA chaperone involved in post-transcriptional regulation that plays a key role in stress response and virulence in diverse bacterial species. To explore whether Hfq-dependent processes might contribute to the regulation of antibiotic production we constructed an S39006 Δhfq mutant. Production of prodigiosin and carbapenem was abolished in this mutant strain, while production of the QS signalling molecule, butanoyl homoserine lactone (BHL), was unaffected. Using transcriptional fusions, we found that Hfq regulates the QS response regulators, SmaR and CarR. Additionally, exoenzyme production and swimming motility were decreased in a Δhfq mutant, and virulence was attenuated in potato and C. elegans models. These results suggest that an Hfq-dependent pathway is involved in the regulation of virulence and secondary metabolite production in S39006.

Mutations in rpsL that confer streptomycin resistance show pleiotropic effects on virulence and the production of a carbapenem antibiotic in Erwinia carotovora.

Spontaneous streptomycin-resistant derivatives of Erwinia carotovora subsp. carotovora strain ATTn10 were isolated. Sequencing of the rpsL locus (encoding the ribosomal protein S12) showed that each mutant was missense, with a single base change, resulting in the substitution of the wild-type lysine by arginine, threonine or asparagine at codon 43. Phenotypic analyses showed that the rpsL mutants could be segregated into two groups: K43R mutants showed reduced production of the beta-lactam secondary metabolite 1-carbapen-2-em-3 carboxylic acid (Car), but little effect on exoenzyme production or virulence in potato tuber tests. By contrast, the K43N and K43T mutations were pleiotropic, resulting in reduced exoenzyme production and virulence, as well as diminished Car production. The effect on Car production was due to reduced transcription of the quorum-sensing-dependent car biosynthetic genes. The effects of K43N and K43T mutations on Car production were partially alleviated by provision of an excess of the quorum-sensing signalling molecule N-(3-oxohexanoyl)-L-homoserine lactone. Finally, a proteomic analysis of the K43T mutant indicated that the abundance of a subset of intracellular proteins was affected by this rpsL mutation.

Non-heme iron oxygenases generate natural structural diversity in carbapenem antibiotics.

Carbapenems are a clinically important antibiotic family. More than 50 naturally occurring carbapenam/ems are known and are distinguished primarily by their C-2/C-6 side chains where many are only differentiated by the oxidation states of these substituents. With a limited palette of variations the carbapenem family comprises a natural combinatorial library, and C-2/C-6 oxidation is associated with increased efficacy. We demonstrate that ThnG and ThnQ encoded by the thienamycin gene cluster in Streptomyces cattleya oxidize the C-2 and C-6 moieties of carbapenems, respectively. ThnQ stereospecifically hydroxylates PS-5 (5) giving N-acetyl thienamycin (2). ThnG catalyzes sequential desaturation and sulfoxidation of PS-5 (5), giving PS-7 (7) and its sulfoxide (9). The enzymes are relatively substrate selective but are proposed to give rise to the oxidative diversity of carbapenems produced by S. cattleya, and orthologues likely function similarly in allied streptomyces. Elucidating the roles of ThnG and ThnQ will focus further investigations of carbapenem antibiotic biosynthesis.

A conserved lysine in beta-lactam synthetase assists ring cyclization: Implications for clavam and carbapenem biosynthesis.

beta-Lactam synthetase (beta-LS) is the paradigm of a growing class of enzymes that form the critical beta-lactam ring in the clavam and carbapenem antibiotics. beta-LS catalyzes a two-stage reaction in which N(2)-(2-carboxyethyl)-L-arginine is first adenylated, and then undergoes intramolecular ring closure. It was previously shown that the forward kinetic commitment to beta-lactam formation is high, and that the overall rate of reaction is partially limited to a protein conformational change rather than to the chemical step alone of closing the strained ring. beta-Lactam formation was evaluated on the basis of X-ray crystal structures, site-specific mutation, and kinetic and computational studies. The combined evidence clearly points to a reaction coordinate involving the formation of a tetrahedral transition state/intermediate stabilized by a conserved Lys. The combination of substrate preorganization, a well-stabilized transition state and an excellent leaving group facilitates this acyl substitution to account for the strong forward commitment to catalysis and to lower the barrier of four-membered ring formation to the magnitude of a protein conformational change.

Synthesis of regio- and stereoselectively deuterium-labelled derivatives of L-glutamate semialdehyde for studies on carbapenem biosynthesis.

L-glutamate semialdehyde (L-GSA) is an intermediate in biosynthetic pathways including those leading to the carbapenem antibiotics. We describe studies on asymmetric deuteration or hydrogenation of appropriate didehydro-amino acid precursors for the stereoselective synthesis of C-2- and/or C-3-[2H]-labelled L-GSA suitable for use in mechanistic studies. Regioselective deuterium incorporation into the 5-position of L-GSA was achieved using a labelled form of the Schwartz reagent (Cp2Zr2HCl). 4,4-Dideuterated and fully backbone deuterated L-GSAs were prepared. The application of the labelled L-GSA derivatives to biosynthetic studies was exemplified by the chemo-enzymatic preparation of selectively deuterated trans-carboxymethylprolines using two different carboxymethylproline synthases (CarB and ThnE), enzymes that catalyse early steps in the biosynthesis of two carbapenems: (5R)-carbapenem-3-carboxylate and thienamycin, respectively.

PigZ, a TetR/AcrR family repressor, modulates secondary metabolism via the expression of a putative four-component resistance-nodulation-cell-division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006.

The Gram-negative enterobacterium, Serratia sp. ATCC 39006 synthesizes several secondary metabolites, including prodigiosin (Pig) and a carbapenem antibiotic (Car). A complex hierarchical network of regulatory proteins control Pig and Car production. In this study we characterize a TetR family regulator, PigZ, which represses transcription of a divergently transcribed putative resistance-nodulation-cell-division (RND) efflux pump, encoded by zrp (PigZ repressed pump) ADBC, via direct binding to the zrpA-pigZ intergenic region. Unusually, this putative RND pump contains two predicted membrane fusion proteins (MFPs), ZrpA and ZrpD. A mutation in pigZ resulted in multiple phenotypic changes, including exoenzyme production, motility and differential regulation of Pig and Car production. A polar suppressor mutation, within zrpA, restored all tested phenotypes to parental strain levels, indicating that the changes observed are due to the increase in expression of ZrpADBC in the absence of the repressor, PigZ. Genomic deletions of zrpA and zrpD indicate that the MFP ZrpD, but not ZrpA, is essential for activity of the putative pump. Bioinformatic analysis revealed that putative RND efflux pumps encoding two MFP components are not uncommon, particularly among plant-associated, Gram-negative bacteria. In addition, based on phylogenetic analysis, we propose that these pairs of MFPs consist of two distinct subtypes.