Discovery and characterization of genes conferring natural resistance to the antituberculosis antibiotic capreomycin.

Metagenomic-based studies have predicted an extraordinary number of potential antibiotic-resistance genes (ARGs). These ARGs are hidden in various environmental bacteria and may become a latent crisis for antibiotic therapy via horizontal gene transfer. In this study, we focus on a resistance gene cph, which encodes a phosphotransferase (Cph) that confers resistance to the antituberculosis drug capreomycin (CMN). Sequence Similarity Network (SSN) analysis classified 353 Cph homologues into five major clusters, where the proteins in cluster I were found in a broad range of actinobacteria. We examine the function and antibiotics targeted by three putative resistance proteins in cluster I via biochemical and protein structural analysis. Our findings reveal that these three proteins in cluster I confer resistance to CMN, highlighting an important aspect of CMN resistance within this gene family. This study contributes towards understanding the sequence-structure-function relationships of the phosphorylation resistance genes that confer resistance to CMN.

Crystal structure of CmnB involved in the biosynthesis of the nonproteinogenic amino acid L-2,3-diaminopropionic acid.

L-2,3-Diaminopropionic acid (L-Dap) is a nonproteinogenic amino acid that plays as an important role as a building block in the biosynthesis of several natural products, including capreomycin, viomycin, zwittermicin, staphyloferrin and dapdiamide. A previous study reported that CmnB and CmnK are two enzymes that are involved in the formation of L-Dap in the biosynthesis of capreomycin. CmnB catalyzes the condensation reaction of O-phospho-L-serine and L-glutamic acid to generate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid, which subsequently undergoes oxidative hydrolysis via CmnK to generate the product L-Dap. Here, the crystal structure of CmnB in complex with the reaction intermediate PLP-α-aminoacrylate is reported at 2.2 Šresolution. Notably, CmnB is the second known example of a PLP-dependent enzyme that forms a monomeric structure in crystal packing. The crystal structure of CmnB also provides insights into the catalytic mechanism of the enzyme and supports the biosynthetic pathway of L-Dap reported in previous studies.

N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry.

Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N-O or O-S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.

Engineering a High-Affinity Anti-Methoxy Poly(ethylene glycol) (mPEG) Antibody for Sensitive Immunosensing of mPEGylated Therapeutics and mPEG Molecules.

Sensitive quantification of methoxy poly(ethylene glycol) (mPEG)-conjugated therapeutics for pharmacokinetic determination is critical for mPEGylated drug development. However, sensitive measurement of low-molecular-weight (lmw) mPEG compounds remains challenging due to epitope competition between backbone-specific anti-PEG antibodies. Here, we engineered a high-affinity methoxy-specific anti-mPEG antibody for sensitive quantification of free mPEG molecules and mPEGylated therapeutics. The affinity-enhanced h15-2Y antibody variant shows a 10.3-fold increase in mPEG-binding activity compared to parental h15-2b. h15-2Y-based sandwich ELISA can effectively quantify lmw mPEG5K and high-molecular-weight (hmw) mPEG20K at concentrations as low as 3.4 and 5.1 ng mL-1, respectively. Moreover, lmw mPEG compounds (560, 750, 1000, and 2000 Da) can be efficiently quantified via h15-2Y-based competitive ELISA with detection limits at nanomolar levels. This study provides a promising approach for application in the quantitative analysis of the various sizes of mPEG molecules to accelerate the timeline of mPEG-conjugated drug development.

Characterization and Structural Determination of CmnG-A, the Adenylation Domain That Activates the Nonproteinogenic Amino Acid Capreomycidine in Capreomycin Biosynthesis.

Capreomycidine (Cap) is a nonproteinogenic amino acid and building block of nonribosomal peptide (NRP) natural products. We report the formation and activation of Cap in capreomycin biosynthesis. CmnC and CmnD catalyzed hydroxylation and cyclization, respectively, of l-Arg to form l-Cap. l-Cap is then adenylated by CmnG-A before being incorporated into the nonribosomal peptide. The co-crystal structures of CmnG-A with l-Cap and adenosine nucleotides provide insights into the specificity and engineering opportunities of this unique adenylation domain.

Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer.

CmnC is an α-ketoglutarate (α-KG)-dependent non-heme iron oxygenase involved in the formation of the l-capreomycidine (l-Cap) moiety in capreomycin (CMN) biosynthesis. CmnC and its homologues, VioC in viomycin (VIO) biosynthesis and OrfP in streptothricin (STT) biosynthesis, catalyze hydroxylation of l-Arg to form ß-hydroxy l-Arg (CmnC and VioC) or ß,γ-dihydroxy l-Arg (OrfP). In this study, a combination of biochemical characterization and structural determination was performed to understand the substrate binding environment and substrate specificity of CmnC. Interestingly, despite having a high conservation of the substrate binding environment among CmnC, VioC, and OrfP, only OrfP can hydroxylate the substrate enantiomer d-Arg. Superposition of the structures of CmnC, VioC, and OrfP revealed a similar folds and overall structures. The active site residues of CmnC, VioC, and OrfP are almost conserved; however Leu136, Ser138, and Asp249 around the substrate binding pocket in CmnC are replaced by Gln, Gly, and Tyr in OrfP, respectively. These residues may play important roles for the substrate binding. The mutagenesis analysis revealed that the triple mutant CmnCL136Q,S138G,D249Y switches the substrate stereoselectivity from l-Arg to d-Arg with ∼6% relative activity. The crystal structure of CmnCL136Q,S138G,D249Y in complex with d-Arg revealed that the substrate loses partial interactions and adopts a different orientation in the binding site. This study provides insights into the enzyme engineering to α-KG non-heme iron oxygenases for adjustment to the substrate stereoselectivity and development of biocatalysts.

Structural determination of an antibody that specifically recognizes polyethylene glycol with a terminal methoxy group.

Covalent attachment of methoxy poly(ethylene) glycol (mPEG) to therapeutic molecules is widely employed to improve their systemic circulation time and therapeutic efficacy. mPEG, however, can induce anti-PEG antibodies that negatively impact drug therapeutic effects. However, the underlying mechanism for specific binding of antibodies to mPEG remains unclear. Here, we determined the first co-crystal structure of the humanized 15-2b anti-mPEG antibody in complex with mPEG, which possesses a deep pocket in the antigen-binding site to accommodate the mPEG polymer. Structural and mutational analyses revealed that mPEG binds to h15-2b via Van der Waals and hydrogen bond interactions, whereas the methoxy group of mPEG is stabilized in a hydrophobic environment between the VH:VL interface. Replacement of the heavy chain hydrophobic V37 residue with a neutral polar serine or threonine residue offers additional hydrogen bond interactions with methoxyl and hydroxyl groups, resulting in cross-reactivity to mPEG and OH-PEG. Our findings provide insights into understanding mPEG-binding specificity and antigenicity of anti-mPEG antibodies.

Biomimetic Total Synthesis of the Spiroindimicin Family of Natural Products.

A unified strategy for the biomimetic total synthesis of the spiroindimicin family of natural products was reported. Key transformations include a one-pot two-enzyme-catalyzed oxidative dimerization of L-tryptophan/5-chloro-L-tryptophan to afford the bis-indole precursors chromopyrrolic acid/5',5''-dichloro-chromopyrrolic acid, and regioselective C3'-C2'' and C3'-C4'' bond formation converting a common bis-indole skeleton to two skeletally different natural products, including (±)-spiroindimicins D and G with a [5,5] spiro-ring skeleton, and (±)-spiroindimicins A and H with a [5,6] spiro-ring skeleton, respectively.

The Structure-Function Relationship of Human Bleomycin Hydrolase: Mutation of a Cysteine Protease into a Serine Protease.

Human bleomycin hydrolase (hBH) catalyzes deamidation of the anticancer drug bleomycins (BLM). This enzyme is involved in BLM detoxification and drug resistance. Herein, we report the putative BLM-binding site and catalytic mechanism of hBH. The crystal structures and biochemical studies suggest that hBH cleaves its C-terminal residue without significant preference for the type of amino acid, and therefore can accordingly accommodate the ß-aminoalanine amide moiety of BLM for deamidation. Interestingly, hBH is capable of switching from a cysteine protease to a serine protease that is unable to cleave the secondary amide of hBH C-terminus but reacts with the primary amide of BLMs.

Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.

Structure-guided product determination of the bacterial type II diterpene synthase Tpn2.

A grand challenge in terpene synthase (TS) enzymology is the ability to predict function from protein sequence. Given the limited number of characterized bacterial TSs and significant sequence diversities between them and their eukaryotic counterparts, this is currently impossible. To contribute towards understanding the sequence-structure-function relationships of type II bacterial TSs, we determined the structure of the terpentedienyl diphosphate synthase Tpn2 from Kitasatospora sp. CB02891 by X-ray crystallography and made structure-guided mutants to probe its mechanism. Substitution of a glycine into a basic residue changed the product preference from the clerodane skeleton to a syn-labdane skeleton, resulting in the first syn-labdane identified from a bacterial TS. Understanding how a single residue can dictate the cyclization pattern in Tpn2, along with detailed bioinformatics analysis of bacterial type II TSs, sets the stage for the investigation of the functional scope of bacterial type II TSs and the discovery of novel bacterial terpenoids.

Engineered photo-chemical therapeutic nanocomposites provide effective antibiofilm and microbicidal activities against bacterial infections in porous devices.

Today, prosthetic joint infection (PJI) is still a relatively rare but devastating complication following total hip and/or knee arthroplasty. The treatment of PJI is difficult due to a number of obstacles, such as microbial drug resistance, biofilm protection, and insufficient immune activity, which dramatically diminish the cure rate of PJI to <50%. To efficiently eradicate the bacteria hiding in the implant, photo-chemical joint antibacterial therapeutics based on indocyanine green (ICG) and rifampicin (RIF) co-loaded PLGA nanoparticles (IRPNPs) were developed in this study. The IRPNPs were first characterized as a spherical nanostructure with a size of 266 ± 18.2 nm and a surface charge of -28 ± 1.6 mV. In comparison with freely dissolved ICG, the IRPNPs may confer enhanced thermal stability to the encapsulated ICG and are able to provide a comparable hyperthermic effect and increased production of singlet oxygen under 808 nm near infrared (NIR) exposure with an intensity of 6 W cm-2. Based on the spectrophotometric analysis, the IRPNPs with ≥20-/3.52 µM ICG/RIF were able to provide remarkable antibiofilm and antimicrobial effects against bacteria in a porous silicon bead upon NIR exposure in vitro. Through the analysis of the microbial population index in an animal study, ≥70% Staphylococcus capitis subsp. urealyticus grown in a porous silicon bead in vivo can be successfully eliminated without organ damage or inflammatory lesions around the implant by using IRPNPs + NIR irradiation every 72 h for 9 days. The resulting bactericidal efficacy was approximately three-fold higher than that resulting from using an equal amount of free RIF alone. Taken together, we anticipate that IRPNP-mediated photochemotherapy can serve as a feasible antibacterial approach for PJI treatment in the clinic.

Characterization of Enzymes Catalyzing the Formation of the Nonproteinogenic Amino Acid l-Dap in Capreomycin Biosynthesis.

Capreomycin (CMN) and viomycin (VIO) are nonribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including l-2,3-diaminopropionic acid (l-Dap), ß-ureidodehydroalanine, l-capreomycidine, and ß-lysine. Previous bioinformatics analysis suggested that CmnB/VioB and CmnK/VioK participate in the formation of l-Dap; however, the real substrates of these enzymes are yet to be confirmed. We herein show that starting from O-phospho-l-Ser (OPS) and l-Glu precursors, CmnB catalyzes the condensation reaction to generate a metabolite intermediate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid (ACEGA), which undergoes NAD+-dependent oxidative hydrolysis by CmnK to generate l-Dap. Furthermore, the binding site of ACEGA and the catalytic mechanism of CmnK were elucidated with the assistance of three crystal structures, including those of apo-CmnK, the NAD+-CmnK complex, and CmnK in an alternative conformation. The CmnK-ACEGA docking model revealed that the glutamate α-hydrogen points toward the nicotinamide moiety. It provides evidence that the reaction is dependent on hydride transfer to form an imine intermediate, which is subsequently hydrolyzed by a water molecule to produce l-Dap. These findings modify the original proposed pathway and provide insights into l-Dap formation in the biosynthesis of other related natural products.

Characterization of TnmH as an O-Methyltransferase Revealing Insights into Tiancimycin Biosynthesis and Enabling a Biocatalytic Strategy To Prepare Antibody-Tiancimycin Conjugates.

The enediynes are among the most cytotoxic molecules known, and their use as anticancer drugs has been successfully demonstrated by targeted delivery. Clinical advancement of the anthraquinone-fused enediynes has been hindered by their low titers and lack of functional groups to enable the preparation of antibody-drug conjugates (ADCs). Here we report biochemical and structural characterization of TnmH from the tiancimycin (TNM) biosynthetic pathway, revealing that (i) TnmH catalyzes regiospecific methylation at the C-7 hydroxyl group, (ii) TnmH exhibits broad substrate promiscuity toward hydroxyanthraquinones and S-alkylated SAM analogues and catalyzes efficient installation of reactive alkyl handles, (iii) the X-ray crystal structure of TnmH provides the molecular basis to account for its broad substrate promiscuity, and (iv) TnmH as a biocatalyst enables the development of novel conjugation strategies to prepare antibody-TNM conjugates. These findings should greatly facilitate the construction and evaluation of antibody-TNM conjugates as next-generation ADCs for targeted chemotherapy.

Pregnenolonyl-α-glucoside exhibits marked anti-cancer and CYP17A1 enzymatic inhibitory activities.

We report here that pregnenolonyl-α-glucoside (2), a steryl glycoside synthesized directly from pregnenolone and glucose via a consecutive multienzyme-catalyzed process, exhibits marked dose-dependent cytotoxic activity against HT29, AGS, and ES-2 cells with IC50 values of 23.5 to 50.9 µM. An in vitro CYP17A1 binding pattern assay and protein-ligand docking model support that 2, like abiraterone, binds in the active site heme iron pocket of CYP17A1.

Terpene synthases in disguise: enzymology, structure, and opportunities of non-canonical terpene synthases.

Covering: up to July 2019 Terpene synthases (TSs) are responsible for generating much of the structural diversity found in the superfamily of terpenoid natural products. These elegant enzymes mediate complex carbocation-based cyclization and rearrangement cascades with a variety of electron-rich linear and cyclic substrates. For decades, two main classes of TSs, divided by how they generate the reaction-triggering initial carbocation, have dominated the field of terpene enzymology. Recently, several novel and unconventional TSs that perform TS-like reactions but do not resemble canonical TSs in sequence or structure have been discovered. In this review, we identify 12 families of non-canonical TSs and examine their sequences, structures, functions, and proposed mechanisms. Nature provides a wide diversity of enzymes, including prenyltransferases, methyltransferases, P450s, and NAD+-dependent dehydrogenases, as well as completely new enzymes, that utilize distinctive reaction mechanisms for TS chemistry. These unique non-canonical TSs provide immense opportunities to understand how nature evolved different tools for terpene biosynthesis by structural and mechanistic characterization while affording new probes for the discovery of novel terpenoid natural products and gene clusters via genome mining. With every new discovery, the dualistic paradigm of TSs is contradicted and the field of terpene chemistry and enzymology continues to expand.

Multiple Pleomorphic Tetramers of Thermostable Direct Hemolysin from Grimontia hollisae in Exerting Hemolysis and Membrane Binding.

Oligomerization of protein into specific quaternary structures plays important biological functions, including regulation of gene expression, enzymes activity, and cell-cell interactions. Here, we report the determination of two crystal structures of the Grimontia hollisae (formally described as Vibrio hollisae) thermostable direct hemolysin (Gh-TDH), a pore-forming toxin. The toxin crystalized in the same space group of P21212, but with two different crystal packing patterns, each revealing three consistent tetrameric oligomerization forms called Oligomer-I, -II, and -III. A central pore with comparable depth of ~50 Å but differing in shape and size was observed in all determined toxin tetrameric oligomers. A common motif of a toxin dimer was found in all determined structures, suggesting a plausible minimum functional unit within the tetrameric structure in cell membrane binding and possible hemolytic activity. Our results show that bacterial toxins may form a single or highly symmetric oligomerization state when exerting their biological functions. The dynamic nature of multiple symmetric oligomers formed upon release of the toxin may open a niche for bacteria survival in harsh living environments.

Genome mining and biosynthesis of kitacinnamycins as a STING activator.

Cinnamoyl-containing nonribosomal peptides (CCNPs) are a small group of secondary metabolites with potent biological activities produced by actinobacteria. Two remarkable features in the biosynthesis of CCNPs include the nonribosomal peptide synthases (NRPSs) for assembly of the depsipeptide backbone and the type II polyketide synthases (PKSs) for N-terminal cinnamoyl moiety construction. Here, we present a genome mining approach targeting both NRPS and type II PKS for discovery of new CCNPs, which led to the identification of 51 putative CCNP gene clusters from public bacterial genome databases. After strain prioritization, a novel class of CCNP-type glycopeptides named kitacinnamycins, one of which showing potent activation ability towards the stimulator of interferon genes (STING) protein, was identified. Bioinformatic, genetic and biochemical analysis revealed the use of the NRPS assembly line to form the macrocyclic peptide backbone, followed by a P450 monooxygenase to generate terminal oxidized groups. A glycosyltransferase with relatively broad substrate specificity transfers sugars to the newly generated OH/COOH group. The protein crystallographic study further provided structural insights into this glycosylation. Our results not only demonstrated the feasibility of genome mining and strain prioritization for the discovery of new bioactive natural products but also disclosed the biosynthetic pathway for kitacinnamycins.

Comparative Studies of the Biosynthetic Gene Clusters for Anthraquinone-Fused Enediynes Shedding Light into the Tailoring Steps of Tiancimycin Biosynthesis.

Comparative analyses of the four known anthraquinone-fused enediynes biosynthetic gene clusters identified four genes, tnmE6, tnmH, tnmL, and tnmQ, unique to the tnm gene cluster. Larger scale fermentation of both the S. sp. CB03234 wild-type and the Δ tnmH and Δ tnmL mutant strains resulted in the characterization of 20 new tiancimycin (TNM) congeners, including five enediynes. These findings enabled a proposal for the late stage of TNM biosynthesis featuring an intermediate possibly common for all anthraquinone-fused enediynes.

Biochemical and Structural Characterization of TtnD, a Prenylated FMN-Dependent Decarboxylase from the Tautomycetin Biosynthetic Pathway.

Tautomycetin (TTN) is a polyketide natural product featuring a terminal alkene. Functional characterization of the genes within the ttn gene cluster from Streptomyces griseochromogenes established the biosynthesis of the TTN polyketide backbone, its dialkylmaleic anhydride moiety, the coupling of the two moieties to form the nascent intermediate TTN F-1, and the tailoring steps converting TTN F-1 to TTN. Here, we report biochemical and structural characterization of TtnD, a prenylated FMN (prFMN)-dependent decarboxylase belonging to the UbiD family that catalyzes the penultimate step of TTN biosynthesis. TtnD catalyzes decarboxylation of TTN D-1 to TTN I-1, utilizing prFMN as a cofactor generated by the TtnC flavin prenyltransferase; both TtnD and TtnC are encoded within the ttn biosynthetic gene cluster. TtnD exhibits substrate promiscuity but accepts only TTN D-1 congeners that feature an α,ß-unsaturated acid, supporting the [3+2] cycloaddition mechanism during catalysis that requires the double bond of an α,ß-unsaturated acid substrate. TtnD shares a similar overall structure with other members of the UbiD family but forms a homotetramer in solution. Each protomer is composed of three domains with the active site located between the middle and C-terminal domains; R169-E272-E277, constituting the catalytic triad, and E228, involved in Mn(II)-mediated binding of prFMN, were confirmed by site-directed mutagenesis. TtnD represents the first example of a prFMN-dependent decarboxylase involved in polyketide biosynthesis, expanding the substrate scope of the UbiD family of decarboxylases beyond simple aromatic and cinnamic acids. TtnD and its homologues are widespread in nature and could be exploited as biocatalysts for organic synthesis.