Hydrolysis of the acetyl-CoA allosteric activator by Staphylococcus aureus pyruvate carboxylase.

Pyruvate carboxylase (PC) catalyzes the carboxylation of pyruvate to oxaloacetate which serves as an important anaplerotic reaction to replenish citric acid cycle intermediates. In most organisms, the PC-catalyzed reaction is allosterically activated by acetyl-coenzyme A. It has previously been reported that vertebrate PC can catalyze the hydrolysis of acetyl-CoA, offering a potential means for the enzyme to attenuate its allosteric activation.

Purification of heteromeric acetyl-CoA carboxylases from Escherichia coli for structure solution.

The primary role of acetyl-CoA carboxylases (ACCs) is to generate malonyl-CoA for use in fatty acid and lipid biosynthesis. However, malonyl-CoA is also used in other various metabolic processes such as secondary metabolite biosynthesis. The diverse utilization of malonyl-CoA makes ACCs targets for the development of inhibitors and also a target for engineering allosteric regulation for biofuel and secondary metabolite production.

Kinetic characterization of the C-terminal domain of Malonyl-CoA reductase.

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains.

Active heteromeric acetyl-CoA carboxylase forms polymorphic helical tubular filaments.

The heteromeric acetyl-CoA carboxylase (ACC) has four subunits assumed to form an elusive catalytic complex and are involved in allosteric and transcriptional regulation. The ACC represents almost all ACCs from pathogenic bacteria making it a key antibiotic development target to fight growing antibiotic resistance. Furthermore, it is a model for cyanobacterial and plant plastid ACCs as biofuel engineering targets.

CAND1 inhibits Cullin-2-RING ubiquitin ligases for enhanced substrate specificity.

Through targeting essential cellular regulators for ubiquitination and serving as a major platform for discovering proteolysis-targeting chimera (PROTAC) drugs, Cullin-2 (CUL2)-RING ubiquitin ligases (CRL2s) comprise an important family of CRLs. The founding members of CRLs, the CUL1-based CRL1s, are known to be activated by CAND1, which exchanges the variable substrate receptors associated with the common CUL1 core and promotes the dynamic assembly of CRL1s. Here we find that CAND1 inhibits CRL2-mediated protein degradation in human cells.

Allosteric Site at the Biotin Carboxylase Dimer Interface Mediates Activation and Inhibition in Pyruvate Carboxylase.

Allosteric regulation of the essential anaplerotic enzyme, pyruvate carboxylase (PC), is vital for metabolic homeostasis. PC catalyzes the bicarbonate- and ATP-dependent carboxylation of pyruvate to form oxaloacetate. Dysregulation of PC activity can impact glucose and redox metabolism, which contributes to the pathogenicity of many diseases.

Structures of chloramphenicol acetyltransferase III and Escherichia coli β-ketoacylsynthase III co-crystallized with partially hydrolysed acetyl-oxa(dethia)CoA.

Acetyl coenzyme A (acetyl-CoA) is a reactive metabolite that nonproductively hydrolyzes in a number of enzyme active sites in the crystallization time frame. In order to elucidate the enzyme-acetyl-CoA interactions leading to catalysis, acetyl-CoA substrate analogs are needed. One possible analog for use in structural studies is acetyl-oxa(dethia)CoA (AcOCoA), in which the thioester S atom of CoA is replaced by an O atom.

Activity of Fatty Acid Biosynthesis Initiating Ketosynthase FabH with Acetyl/Malonyl-oxa/aza(dethia)CoAs.

Fatty acid and polyketide biosynthetic enzymes exploit the reactivity of acyl- and malonyl-thioesters for catalysis. A prime example is FabH, which initiates fatty acid biosynthesis in many bacteria and plants. FabH performs an acyltransferase reaction with acetyl-CoA to generate an acetyl--FabH acyl-enzyme intermediate and subsequent decarboxylative Claisen-condensation with a malonyl-thioester carried by an acyl carrier protein (ACP).

Structure-Function Studies of Two Yeast Homing Endonucleases that Evolved to Cleave Identical Targets with Dissimilar Rates and Specificities.

The LAGLIDADG family of homing endonucleases (LHEs) bind to and cleave their DNA recognition sequences with high specificity. Much of our understanding for how these proteins evolve their specificities has come from studying LHE homologues. To gain insight into the molecular basis of LHE specificity, we characterized I-WcaI, the homologue of the Saccharomyces cerevisiae I-SceI LHE found in Wickerhamomyces canadensis.

Substrate Enolate Intermediate and Mimic Captured in the Active Site of Streptomyces coelicolor Methylmalonyl-CoA Epimerase.

Methylmalonyl-CoA epimerase (MMCE) is proposed to use general acid-base catalysis, but the proposed catalytic glutamic acids are highly asymmetrical in the active site unlike many other racemases. To gain insight into the puzzling relationships between catalytic mechanism, structure, and substrate preference, we solved Streptomyces coelicolor MMCE structures with substrate or 2-nitropropionyl-CoA, an intermediate/transition state analogue. Both ligand bound structures have a planar methylmalonate/2-nitropropionyl moiety indicating a deprotonated C2 with ≥4 Å distances to either catalytic acid.

Structures of LnmK, a Bifunctional Acyltransferase/Decarboxylase, with Substrate Analogues Reveal the Basis for Selectivity and Stereospecificity.

LnmK stereospecifically accepts (2)-methylmalonyl-CoA, generating propionyl--acyl carrier protein to support polyketide biosynthesis. LnmK and its homologues are the only known enzymes that carry out a decarboxylation (DC) and acyl transfer (AT) reaction in the same active site as revealed by structure-function studies. Substrate-assisted catalysis powers LnmK, as decarboxylation of (2)-methylmalonyl-CoA generates an enolate capable of deprotonating active site Tyr62, and the Tyr62 phenolate subsequently attacks propionyl-CoA leading to a propionyl--LnmK acyl-enzyme intermediate.

The LnmK Bifunctional Acyltransferase/Decarboxylase Specifying (2)-Methylmalonyl-CoA and Employing Substrate-Assisted Catalysis for Polyketide Biosynthesis.

We previously showed that the bifunctional LnmK acyltransferase/decarboxylase (AT/DC) catalyzed the formation of a propionyl--acyl carrier protein (ACP) from methylmalonyl-CoA, but its substrate specificity to (2)-, (2)-, or (2)-methylmalonyl CoA was not known. We subsequently revealed that LnmK AT and DC activities share the same active site, employing a Tyr as the catalytic residue for AT, but failed to identify a general base within the vicinity of the active site for LnmK catalysis. We now show that (i) LnmK specifies (2)-methylmalonyl-CoA and (ii) the AT and DC activities are coupled, featuring substrate-assisted catalysis via the enolate to account for the missing general base within the LnmK active site.

Vitamin C and B as new biomaterials to alter intestinal stem cells.

Vitamin C (ascorbic acid) and vitamin B (niacin) have been extensively studied since the 20th century. In the area of stem cell biology, vitamin C has shown its direct impact toward homeostasis and epigenetic changes (D'Aniello et al., Stem Cells International, 2017, 1-16).

Sulfonate/Nitro Bearing Methylmalonyl-Thioester Isosteres Applied to Methylmalonyl-CoA Decarboxylase Structure-Function Studies.

Malonyl-thioesters are reactive centers of malonyl-CoA and malonyl- S-acyl carrier protein, essential to fatty acid, polyketide and various specialized metabolite biosynthesis. Enzymes that create or use malonyl-thioesters spontaneously hydrolyze or decarboxylate reactants on the crystallographic time frame preventing determination of structure-function relationships. To address this problem, we have synthesized a panel of methylmalonyl-CoA analogs with the carboxylate represented by a sulfonate or nitro and the thioester retained or represented by an ester or amide.

P450-Catalyzed Tailoring Steps in Leinamycin Biosynthesis Featuring Regio- and Stereoselective Hydroxylations and Substrate Promiscuities.

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140. Both in vivo and in vitro characterization of the LNM biosynthetic machinery have established the formation of the 18-membered macrolactam backbone and the C-3 alkyl branch; the nascent product, LNM E1, of the hybrid nonribosomal peptide synthetase (NRPS)-acyltransferase (AT)-less type I polyketide synthase (PKS); and the generation of the thiol moiety at C-3 of LNM E1. However, the tailoring steps converting LNM E1 to LNM are still unknown.

Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027.

C-1027 is a chromoprotein enediyne antitumor antibiotic, consisting of the CagA apoprotein and the C-1027 chromophore. The C-1027 chromophore features a nine-membered enediyne core appended with three peripheral moieties, including an ( S)-3-chloro-5-hydroxy-β-tyrosine. In a convergent biosynthesis of the C-1027 chromophore, the ( S)-3-chloro-5-hydroxy-β-tyrosine moiety is appended to the enediyne core by the free-standing condensation enzyme SgcC5.

PokMT1 from the Polyketomycin Biosynthetic Machinery of Streptomyces diastatochromogenes Tü6028 Belongs to the Emerging Family of C-Methyltransferases That Act on CoA-Activated Aromatic Substrates.

Recent biochemical characterizations of the MdpB2 CoA ligase and MdpB1 C-methyltransferase (C-MT) from the maduropeptin (MDP, 2) biosynthetic machinery revealed unusual pathway logic involving C-methylation occurring on a CoA-activated aromatic substrate. Here we confirmed this pathway logic for the biosynthesis of polyketomycin (POK, 3). Biochemical characterization unambiguously established that PokM3 and PokMT1 catalyze the sequential conversion of 6-methylsalicylic acid (6-MSA, 4) to form 3,6-dimethylsalicylyl-CoA (3,6-DMSA-CoA, 6), which serves as the direct precursor for the 3,6-dimethylsalicylic acid (3,6-DMSA) moiety in the biosynthesis of 3.

Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis.

Enediynes are potent natural product anticancer antibiotics, and are classified as 9- or 10-membered according to the size of their enediyne core carbon skeleton. Both 9- and 10-membered enediyne cores are biosynthesized by the enediyne polyketide synthase (PKSE), thioesterase (TE), and PKSE-associated enzymes. Although the divergence between 9- and 10-membered enediyne core biosynthesis remains unclear, it has been observed that nascent polyketide intermediates, tethered to the acyl carrier protein (ACP) domain of PKSE, could be released by TE in the absence of the PKSE-associated enzymes.

Strain Prioritization and Genome Mining for Enediyne Natural Products.

Unlabelled: The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster.

Hemoglobin extravasation in the brain of rats exchange-transfused with hemoglobin-based oxygen carriers.

Haemoglobin (Hb)-based oxygen carriers are under consideration as oxygen therapeutics. Their effect on apoptosis is critical, because the onset of pro-apoptotic pathways may lead to tissue damage. MP4OX, a polyethylene glycol-conjugated human Hb preserves the baseline level of neuron apoptosis with respect to sham.

Dissolution dynamic nuclear polarization of deuterated molecules enhanced by cross-polarization.

We present novel means to hyperpolarize deuterium nuclei in CD groups at cryogenic temperatures. The method is based on cross-polarization from H to C and does not require any radio-frequency fields applied to the deuterium nuclei. After rapid dissolution, a new class of long-lived spin states can be detected indirectly by C NMR in solution.

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027.

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus.

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier Protein-Tethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus.

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-β-tyrosine. This two-component monooxygenase is remarkable for two reasons.

Discovery of (R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one (AMG 337), a Potent and Selective Inhibitor of MET with High Unbound Target Coverage and Robust In Vivo Antitumor Activity.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.