Biosynthesis of isonitrile lipopeptides.

Isonitrile lipopeptides discovered from Actinobacteria have attracted wide attention due to their fascinating biosynthetic pathways and relevance to the virulence of many human pathogens including Mycobacterium tuberculosis. Specifically, the identification of the new class of isonitrile-forming enzymes that belong to non-heme iron (II) and α-ketoglutarate dependent dioxygenases has intrigued several research groups to investigate their catalytic mechanism. Here we summarize the recent studies on the biosynthesis of isonitrile lipopeptides from Streptomyces and Mycobacterium. The latest research on the core and tailoring enzymes involved in the pathway as well as the isonitrile metabolic enzymes are discussed in this review.

Abi Family Protein, DidK, Is Involved in the Maturation of Anticancer Depsipeptide, Didemnin B.

Didemnin B is a marine-derived depsipeptide with potent antiviral and anticancer activities. A prodrug activation mechanism was previously proposed for the biosynthesis of didemnin B by the nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly line, but the enzyme involved in the maturation process remained unknown. Herein, we demonstrated that DidA, a dimodular NRPS predicted with unrelated activity to didemnin B structure assembly, was indispensable to produce didemnin B, confirming the prodrug mechanism in didemnin B biosynthesis. We further identified an Abi family transmembrane protease, DidK, that functioned as an esterase in the maturation step of didemnin B by in vivo gene knockout and heterologous expression. DidK is structurally distinct from other known hydrolytic enzymes involved in the maturation of bacterial nonribosomal peptides and is the first Abi family protein known to participate in NRPS/PKS-derived natural product production. Further bioinformatic analysis revealed more than 20 DidK homologues encoded in bacterial NRPS/PKS BGCs, suggesting that the involvement of Abi family proteins in natural product biosynthesis might not be rare. These results not only clarify the priming and maturation steps of didemnin B biosynthesis but also expand the function scope of Abi family proteins.

Design and characterization of PROTAC degraders specific to protein N-terminal methyltransferase 1.

Protein N-terminal methylation catalyzed by N-terminal methyltransferase 1 (NTMT1) is an emerging methylation present in eukaryotes, playing important regulatory roles in various biological and cellular processes. Although dysregulation of NTMT1 has been linked to many diseases such as colorectal cancer, their molecular and cellular mechanisms remain elusive due to inaccessibility to an effective cellular probe. Here we report the design, synthesis, and characterization of the first-in-class NTMT1 degraders based on proteolysis-targeting chimera (PROTAC) strategy. Through a brief structure-activity relationship (SAR) study of linker length, a cell permeable degrader 1 involving a von Hippel-Lindau (VHL) E3 ligase ligand was developed and demonstrated to reduce NTMT1 protein levels effectively and selectively in time- and dose-dependent manners in colorectal carcinoma cell lines HCT116 and HT29. Degrader 1 displayed DC50 = 7.53 µM and Dmax > 90% in HCT116 (cellular IC50 > 100 µM for its parent inhibitor DC541). While degrader 1 had marginal cytotoxicity, it displayed anti-proliferative activity in 2D and 3D culture environment, resulting from cell cycle arrested at G0/G1 phase in HCT116. Label-free global proteomic analysis revealed that degrader 1 induced overexpression of calreticulin (CALR), an immunogenic cell death (ICD) signal protein that is known to elicit antitumor immune response and clinically linked to a high survival rate of patients with colorectal cancer upon its upregulation. Collectively, degrader 1 offers the first selective cellular probe for NTMT1 exploration and a new drug discovery modality for NTMT1-related oncology and diseases.

In vivo methylation of OLA1 revealed by activity-based target profiling of NTMT1.

N-Terminal methyltransferase 1 (NTMT1) catalyzes the N-terminal methylation of proteins with a specific N-terminal motif after methionine removal. Aberrant N-terminal methylation has been implicated in several cancers and developmental diseases. Together with motif sequence and signal peptide analyses, activity-based substrate profiling of NTMT1 utilizing (E)-hex-2-en-5-ynyl-S-adenosyl-l-methionine (Hey-SAM) revealed 72 potential targets, which include several previously confirmed ones and many unknowns. Target validation using normal and NTMT1 knock-out (KO) HEK293FT cells generated by CRISPR-Cas9 demonstrated that Obg-like ATPase 1 (OLA1), a protein involved in many critical cellular functions, is methylated in vivo by NTMT1. Additionally, Hey-SAM synthesis achieved ≥98% yield for SAH conversion.

Enantioselective Synthesis of Dilignol Model Compounds and Their Stereodiscrimination Study with a Dye-Decolorizing Peroxidase.

A four-step enantioselective approach was developed to synthesize anti (1R,2S)-1a and (1S,2R)-1b containing a ß-O-4 linkage in good yields. A significant difference was observed for the apparent binding affinities of four stereospecific lignin model compounds with TcDyP by surface plasmon resonance, which was not translated into a significant difference in enzyme activities. The discrepancy may be attributed to the conformational change involving a loop widely present in DyPs upon H2O2 binding.

Pigs immunized with a novel E2 subunit vaccine are protected from subgenotype heterologous classical swine fever virus challenge.

BACKGROUND: Classical swine fever (CSF) or hog cholera is a highly contagious swine viral disease. CSF endemic countries have to use routine vaccination with modified live virus (MLV) vaccines to prevent and control CSF. However, it is impossible to serologically differentiate MLV vaccinated pigs from those infected with CSF virus (CSFV). The aim of this study is to develop a one-dose E2-subunit vaccine that can provide protection against CSFV challenge. We hypothesize that a vaccine consisting of a suitable adjuvant and recombinant E2 with natural conformation may induce a similar level of protection as the MLV vaccine. RESULTS: Our experimental vaccine KNB-E2 was formulated with the recombinant E2 protein (Genotype 1.1) expressed by insect cells and an oil-in-water emulsion based adjuvant. 10 pigs (3 weeks old, 5 pigs/group) were immunized intramuscularly with one dose or two doses (3 weeks apart) KNB-E2, and 10 more control pigs were administered normal saline solution only. Two weeks after the second vaccination, all KNB-E2 vaccinated pigs and 5 control pigs were challenged with 5 × 10(5) TCID50 CSFV Honduras/1997 (Genotype 1.3, 1 ml intramuscular, 1 ml intranasal). It was found that while control pigs infected with CSFV stopped growing and developed high fever (>40 °C), high level CSFV load in blood and nasal fluid, and severe leukopenia 3-14 days post challenge, all KNB-E2 vaccinated pigs continued to grow as control pigs without CSFV exposure, did not show any fever, had low or undetectable level of CSFV in blood and nasal fluid. At the time of CSFV challenge, only pigs immunized with KNB-E2 developed high levels of E2-specific antibodies and anti-CSFV neutralizing antibodies. CONCLUSIONS: Our studies provide direct evidence that pigs immunized with one dose KNB-E2 can be protected clinically from CSFV challenge. This protection is likely mediated by high levels of E2-specific and anti-CSFV neutralizing antibodies.

Study of Class I and Class III Polyhydroxyalkanoate (PHA) Synthases with Substrates Containing a Modified Side Chain.

Polyhydroxyalkanoates (PHAs) are carbon and energy storage polymers produced by a variety of microbial organisms under nutrient-limited conditions. They have been considered as an environmentally friendly alternative to oil-based plastics due to their renewability, versatility, and biodegradability. PHA synthase (PhaC) plays a central role in PHA biosynthesis, in which its activity and substrate specificity are major factors in determining the productivity and properties of the produced polymers. However, the effects of modifying the substrate side chain are not well understood because of the difficulty to accessing the desired analogues. In this report, a series of 3-(R)-hydroxyacyl coenzyme A (HACoA) analogues were synthesized and tested with class I synthases from Chromobacterium sp. USM2 (PhaCCs and A479S-PhaCCs) and Caulobacter crescentus (PhaCCc) as well as class III synthase from Allochromatium vinosum (PhaECAv). It was found that, while different PHA synthases displayed distinct preference with regard to the length of the alkyl side chains, they could withstand moderate side chain modifications such as terminal unsaturated bonds and the azide group. Specifically, the specific activity of PhaCCs toward propynyl analogue (HHxyCoA) was only 5-fold less than that toward the classical substrate HBCoA. The catalytic efficiency (kcat/Km) of PhaECAv toward azide analogue (HABCoA) was determined to be 2.86 × 10(5) M(-1) s(-1), which was 6.2% of the value of HBCoA (4.62 × 10(6) M(-1) s(-1)) measured in the presence of bovine serum albumin (BSA). These side chain modifications may be employed to introduce new material functions to PHAs as well as to study PHA biogenesis via click-chemistry, in which the latter remains unknown and is important for metabolic engineering to produce PHAs economically.

Characterization of Dye-decolorizing Peroxidase (DyP) from Thermomonospora curvata Reveals Unique Catalytic Properties of A-type DyPs.

Dye-decolorizing peroxidases (DyPs) comprise a new family of heme peroxidases, which has received much attention due to their potential applications in lignin degradation. A new DyP from Thermomonospora curvata (TcDyP) was identified and characterized. Unlike other A-type enzymes, TcDyP is highly active toward a wide range of substrates including model lignin compounds, in which the catalytic efficiency with ABTS (kcat(app)/Km(app) = (1.7 × 10(7)) m(-1) s(-1)) is close to that of fungal DyPs. Stopped-flow spectroscopy was employed to elucidate the transient intermediates as well as the catalytic cycle involving wild-type (wt) and mutant TcDyPs. Although residues Asp(220) and Arg(327) are found necessary for compound I formation, His(312) is proposed to play roles in compound II reduction. Transient kinetics of hydroquinone (HQ) oxidation by wt-TcDyP showed that conversion of the compound II to resting state is a rate-limiting step, which will explain the contradictory observation made with the aspartate mutants of A-type DyPs. Moreover, replacement of His(312) and Arg(327) has significant effects on the oligomerization and redox potential (E°') of the enzyme. Both mutants were found to promote the formation of dimeric state and to shift E°' to a more negative potential. Not only do these results reveal the unique catalytic property of the A-type DyPs, but they will also facilitate the development of these enzymes as lignin degraders.