Crystal structure of ClA1, a type of chlorinase from soil bacteria.

Halogenated compounds are widely discovered in nature, and many of them exhibit biological activities, such as an important chlorinated natural product salinosporamide A serving as a potential anticancer agent. Compared with bromination, iodination and fluorination, chlorination is the mainly important modification. To shed light on the mechanism of SAM-dependent chlorinases, a recombinant chlorinase ClA1 was expressed in Escherichia coli and further purified for crystallization and X-ray diffraction experiments. The flake crystals of ClA1 were able to diffract to a resolution of 1.85 Å. The crystals belonged to space group R3, with unit-cell parameters α = ß = 90.0°, γ = 120.0°. By determining the structure of ClA1, it is revealed that the side chain of Arg242 in ClA1 may have contacts with the L-Met. However, in SalL the equivalent Arg243's side chain is far from L-Met. Considering the ClA1 and SalL are from different environments and their enzyme kinetics are quite different, it is suggested that the side chain conformation differences of the conserved arginine are possibly related with the enzyme activity differences of the two chlorinases.

Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates.

Biocatalysis has been increasingly used for pharmaceutical synthesis in an effort to make manufacturing processes greener and more sustainable. Biocatalysts that possess excellent activity, specificity, thermostability and solvent-tolerance are highly sought after to meet the requirements of practical applications. Generating biocatalysts with these specific properties can be achieved by either discovery of novel biocatalysts or protein engineering. Meanwhile, chemoenzymatic routes have also been designed and developed for pharmaceutical synthesis on an industrial scale. This review discusses the recent discoveries, engineering, and applications of biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. Key classes of biocatalysts include reductases, oxidases, hydrolases, lyases, isomerases, and transaminases.

Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non-native Substrate.

Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5'-chloro-5'-deoxyadenosine (5'-ClDA) into 5'-fluoro-5'-deoxyadenosine (5'-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5'-[18 F]FDA, with overall radiochemical conversion (RCC) more than 3-fold higher than wild-type FlA1. Kinetic studies of the two-step reaction revealed that the variants show a significantly improved kcat value in the conversion of 5'-ClDA into S-adenosyl-l-methionine (SAM) but a reduced kcat value in the conversion of SAM into 5'-FDA.

Insights into the programmed ketoreduction of partially reducing polyketide synthases: stereo- and substrate-specificity of the ketoreductase domain.

One of the hallmarks of iterative polyketide synthases (PKSs) is the programming mechanism which is essential for the generation of structurally diverse polyketide products. In partially reducing iterative PKSs (PR-PKSs), the programming mechanism is mainly dictated by the ketoreductase (KR) domain. The KR domain contributes to the programming of PR-PKSs through selective reduction of polyketide intermediates. How the KR domain achieves the selective ketoreduction remains to be fully understood. In this study, we found that the KR domain of the (R)-mellein-synthesizing PR-PKS SACE5532 functions as a B-type KR domain to generate (R)-hydroxyl functionalities. Comparative studies of the KR domains of SACE5532 and NcsB suggested that the two KR domains have distinct substrate preferences towards simple N-acetylcysteamine thioester (SNAC) substrates. We further found that the substrate preference of KRSACE5532 can be switched by swapping several motifs with KRNcsB, and that swapping of the same motifs in the full length SACE5532 resulted in a reprogramming of the PKS. Together, the results advance our understanding of the programming of iterative PR-PKSs by providing new support to the hypothesis that the programmed ketoreduction is accomplished by differential recognition of polyketide intermediates.

Crystal structure of the acyltransferase domain of the iterative polyketide synthase in enediyne biosynthesis.

Biosynthesis of the enediyne natural product dynemicin in Micromonospora chersina is initiated by DynE8, a highly reducing iterative type I polyketide synthase that assembles polyketide intermediates from the acetate units derived solely from malonyl-CoA. To understand the substrate specificity and the evolutionary relationship between the acyltransferase (AT) domains of DynE8, fatty acid synthase, and modular polyketide synthases, we overexpressed a 44-kDa fragment of DynE8 (hereafter named AT(DYN10)) encompassing its entire AT domain and the adjacent linker domain. The crystal structure at 1.4 Å resolution unveils a α/ß hydrolase and a ferredoxin-like subdomain with the Ser-His catalytic dyad located in the cleft between the two subdomains. The linker domain also adopts a α/ß fold abutting the AT catalytic domain. Co-crystallization with malonyl-CoA yielded a malonyl-enzyme covalent complex that most likely represents the acyl-enzyme intermediate. The structure explains the preference for malonyl-CoA with a conserved arginine orienting the carboxylate group of malonate and several nonpolar residues that preclude α-alkyl malonyl-CoA binding. Co-crystallization with acetyl-CoA revealed two noncovalently bound acetates generated by the enzymatic hydrolysis of acetyl-CoA that acts as an inhibitor for DynE8. This suggests that the AT domain can upload the acyl groups from either malonyl-CoA or acetyl-CoA onto the catalytic Ser(651) residue. However, although the malonyl group can be transferred to the acyl carrier protein domain, transfer of the acetyl group to the acyl carrier protein domain is suppressed. Local structural differences may account for the different stability of the acyl-enzyme intermediates.

Rigidifying acyl carrier protein domain in iterative type I PKS CalE8 does not affect its function.

Acyl carrier protein (ACP) domains shuttle acyl intermediates among the catalytic domains of multidomain type I fatty acid synthase and polyketide synthase (PKS) systems. It is believed that the unique function of ACPs is associated with their dynamic property, but it remains to be fully elucidated what type of protein dynamics is critical for the shuttling domain. Using NMR techniques, we found that the ACP domain of iterative type I PKS CalE8 from Micromonospora echinospora is highly dynamic on the millisecond-second timescale. Introduction of an interhelical disulfide linkage in the ACP domain suppresses the dynamics on the millisecond-second timescale and reduces the mobility on the picosecond-nanosecond timescale. We demonstrate that the full-length PKS is fully functional upon rigidification of the ACP domain, suggesting that although the flexibility of the disordered terminal linkers may be important for the function of the ACP domain, the internal dynamics of the helical regions is not critical for that function.

Synthesis of (R)-mellein by a partially reducing iterative polyketide synthase.

Mellein and the related 3,4-dihydroisocoumarins are a family of natural products with interesting biological properties. The mechanisms of dihydroisocoumarin biosynthesis remain largely speculative today. Here we report the synthesis of mellein by a partially reducing iterative polyketide synthase (PR-PKS) as a pentaketide product. Remarkably, despite the head-to-tail homology shared with several fungal and bacterial PR-PKSs, the mellein synthase exhibits a distinct keto reduction pattern in the synthesis of the pentaketide. We present evidence to show that the ketoreductase (KR) domain alone is able to recognize and differentiate the polyketide intermediates, which provides a mechanistic explanation for the programmed keto reduction in these PR-PKSs.

Expression, purification and characterization of the acyl carrier protein phosphodiesterase from Pseudomonas Aeruginosa.

Acyl carrier protein phosphodiesterases (AcpH) are the only enzymes known to remove the 4'-phosphopantetheinyl moiety from holo acyl carrier proteins (ACP), which are a large family of proteins essential for the biosynthesis of lipid and other cellular metabolites. Here we report that the AcpH (paAcpH) from Pseudomonas aeruginosa can be overexpressed in Escherichia coli as a soluble and stable protein after optimization of the expression and purification conditions. This marks an improvement from the aggregation-prone E. coli AcpH that could only be obtained by refolding the polypeptide obtained from the inclusion body. With the soluble recombinant protein, we found that PaAcpH exhibits preferred substrate specificity towards the ACPs from the fatty acid synthesis pathway among eight carrier proteins. We further showed that PaAcpH hydrolyzes and releases the 4'-phosphopantetheinyl group-linked products from a multidomain polyketide synthase, demonstrating that the enzyme is fully capable of hydrolyzing acylated ACP substrates.

A New Biosensor for Stilbenes and a Cannabinoid Enabled by Genome Mining of a Transcriptional Regulator.

In vivo biosensors are powerful tools for metabolic engineering and synthetic biology applications. However, the development of biosensors is hindered by the limited number of characterized transcriptional regulators. The versatile sensing abilities of microbes and genome sequences available hold great potential for developing novel biosensors via genome mining for new transcriptional regulators. Here we report the development and engineering of a new stilbene-responsive biosensor discovered by mining the Novosphingobium aromaticivorans DSM 12444 genome. The biosensor can distinguish resveratrol from its precursors, p-coumaric acid and trans-cinnamic acid. Remarkably, it can detect other biologically active stilbenes with resorcinol groups, and cannabidiolic acid with a ß-resorcylic acid functional group. When coupled to resveratrol biosynthesis enzymes, the biosensor can sense altered resveratrol production in cells, demonstrating a 667-fold enrichment in one round of fluorescence-activated cell sorting. Our biosensor will be potentially applicable to metabolic engineering of microbial cell factories for production of stilbenes and cannabinoids.

Recent advances in combinatorial biosynthesis for drug discovery.

Because of extraordinary structural diversity and broad biological activities, natural products have played a significant role in drug discovery. These therapeutically important secondary metabolites are assembled and modified by dedicated biosynthetic pathways in their host living organisms. Traditionally, chemists have attempted to synthesize natural product analogs that are important sources of new drugs. However, the extraordinary structural complexity of natural products sometimes makes it challenging for traditional chemical synthesis, which usually involves multiple steps, harsh conditions, toxic organic solvents, and byproduct wastes. In contrast, combinatorial biosynthesis exploits substrate promiscuity and employs engineered enzymes and pathways to produce novel "unnatural" natural products, substantially expanding the structural diversity of natural products with potential pharmaceutical value. Thus, combinatorial biosynthesis provides an environmentally friendly way to produce natural product analogs. Efficient expression of the combinatorial biosynthetic pathway in genetically tractable heterologous hosts can increase the titer of the compound, eventually resulting in less expensive drugs. In this review, we will discuss three major strategies for combinatorial biosynthesis: 1) precursor-directed biosynthesis; 2) enzyme-level modification, which includes swapping of the entire domains, modules and subunits, site-specific mutagenesis, and directed evolution; 3) pathway-level recombination. Recent examples of combinatorial biosynthesis employing these strategies will also be highlighted in this review.

Induced-fit upon ligand binding revealed by crystal structures of the hot-dog fold thioesterase in dynemicin biosynthesis.

Dynemicins are structurally related 10-membered enediyne natural products isolated from Micromonospora chernisa with potent antitumor and antibiotic activity. The early biosynthetic steps of the enediyne moiety of dynemicins are catalyzed by an iterative polyketide synthase (DynE8) and a thioesterase (DynE7). Recent studies indicate that the function of DynE7 is to off-load the linear biosynthetic intermediate assembled on DynE8. Here, we report crystal structures of DynE7 in its free form at 2.7 Å resolution and of DynE7 in complex with the DynE8-produced all-trans pentadecen-2-one at 2.1 Å resolution. These crystal structures reveal that upon ligand binding, significant conformational changes throughout the substrate-binding tunnel result in an expanded tunnel that traverses an entire monomer of the tetrameric DynE7 protein. The enlarged inner segment of the channel binds the carbonyl-conjugated polyene mainly through hydrophobic interactions, whereas the putative catalytic residues are located in the outer segment of the channel. The crystallographic information reinforces an unusual catalytic mechanism that involves a strictly conserved arginine residue for this subfamily of hot-dog fold thioesterases, distinct from the typical mechanism for hot-dog fold thioesterases that utilizes an acidic residue for catalysis.

Products of the iterative polyketide synthases in 9- and 10-membered enediyne biosynthesis.

The iterative polyketide synthases from the biosynthetic pathways of three enediyne natural products were examined. The results established the all-trans conjugated pentadecaheptanene as the only major product shared by the PKSs. The experiments further revealed some intrinsic differences among the PKSs by demonstrating the formation of different by-products.

Structure and catalytic mechanism of the thioesterase CalE7 in enediyne biosynthesis.

The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-A crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg(37) plays a crucial role in the hydrolysis of the thioester bond, and that Tyr(29) and a hydrogen-bonded water network assist the decarboxylation of the beta-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4=C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism.

In vitro and in vivo antifungal activities of the eight steroid saponins from Tribulus terrestris L. with potent activity against fluconazole-resistant fungal pathogens.

Antifungal activity of natural products is being studied widely. Saponins are known to be antifungal and antibacterial. We have isolated eight steroid saponins from Tribulus terrestris L., namely TTS-8, TTS-9, TTS-10, TTS-11, TTS-12, TTS-13, TTS-14 and TTS-15. TTS-12 and TTS-15 were identified as tigogenin-3-O-beta-D-xylopyranosyl(1-->2)-[beta-D-xylopyranosyl(1-->3)]-beta-D-glucopyranosyl(1-->4)-[alpha-L-rhamnopyranosyl(1-->2)]-beta-D-galactopyranoside and tigogenin-3-O-beta-D-glucopyranosyl(1-->2)-[beta-D-xylopyranosyl(1-->3)]-beta-D-glucopyranosyl(1-->4)-beta-D-galactopyranoside, respectively. The in vitro antifungal activities of the eight saponins against six fluconazole-resistant yeasts, Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, and Cryptococcus neoformans were studied using microbroth dilution assay. The results showed that TTS-12 and TTS-15 were very effective against several pathogenic candidal species and C. neoformans in vitro. It is noteworthy that TTS-12 and TTS-15 were very active against fluconazole-resistant C. albicans (MIC(80)=4.4, 9.4 microg/ml), C. neoformans (MIC(80)=10.7, 18.7 microg/ml) and inherently resistant C. krusei (MIC(80)=8.8, 18.4 microg/ml). So in vivo activity of TTS-12 in a vaginal infection model with fluconazole-resistant C. albicans was studied in particular. Our studies revealed TTS-12 also showed in vivo activities against fluconazole-resistant yeasts. In conclusion, steroid saponins TTS-12 and TTS-15 from Tribulus terrestris L. have significant in vitro antifungal activity against fluconazole-resistant fungi, especially TTS-12 also showed in vivo activity against fluconazole-resistant C. albicans.