The ASPREE Healthy Ageing Biobank: Methodology and participant characteristics.

ASPirin in Reducing Events in the Elderly (ASPREE), a placebo-controlled prevention trial of low dose aspirin, provided the opportunity to establish a biospecimen biobank from initially healthy persons aged 70+ years for future research. The ASPREE Healthy Ageing Biobank (ASPREE Biobank) collected, processed and stored blood and urine samples at -80degC or under nitrogen vapour at two timepoints, three years apart, from a willing subset of Australian ASPREE participants. Written informed consent included separate opt-in questions for biomarker and genetic testing.

Functional Crosstalk between Discrete Indole Terpenoid Gene Clusters in .

Indole terpenoids make up a large group of secondary metabolites that display an enticing array of bioactivities. While indole diterpene (IDT) and rarely indole sesquiterpene (IST) pathways have been found individually in filamentous fungi, here we show that both cluster types are encoded within the genome of . Through heterologous reconstruction, we demonstrate the cluster encodes for IST biosynthesis and can tailor IDT substrates produced by the cluster.

Incorporation of SARS-CoV-2 spike NTD to RBD protein vaccine improves immunity against viral variants.

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein.

Generation of Alternate Indole Diterpene Architectures in Two Species of .

The significant structural diversity and potent bioactivity of the fungal indole diterpenes (IDTs) has attracted considerable interest in their biosynthesis. Although substantial skeletal diversity is generated by the action of noncanonical terpene cyclases, comparatively little is known about these enzymes, particularly those involved in the generation of the subgroup containing emindole SA and DA, which show alternate terpenoid skeletons. Here, we describe the IDT biosynthetic machinery generating these unusual IDT architectures from and .

Structure, mechanism and inhibition of anthranilate phosphoribosyltransferase.

Anthranilate phosphoribosyltransferase catalyses the second reaction in the biosynthesis of tryptophan from chorismate in microorganisms and plants. The enzyme is homodimeric with the active site located in the hinge region between two domains. A range of structures in complex with the substrates, substrate analogues and inhibitors have been determined, and these have provided insights into the catalytic mechanism of this enzyme.

Reactivity and mechanism in chemical and synthetic biology.

Physical organic chemistry and mechanistic thinking provide a strong intellectual framework for understanding the chemical logic of evolvable informational macromolecules and metabolic transformations in living organisms. These concepts have also led to numerous successes in designing and applying tools to delineate biological function in health and disease, chemical ecology and possible alternative chemistries employed by extraterrestrial life. A symposium at the 2020 Pacifichem meeting was scheduled in December 2020 to discuss designing and exploiting expanded genetic alphabets, methods to understand the biosynthesis of natural products and re-engineering primary metabolism in bacteria.

Conformational interdomain flexibility in a bacterial α-isopropylmalate synthase is necessary for leucine biosynthesis.

α-Isopropylmalate synthase (IPMS) catalyzes the first step in leucine (Leu) biosynthesis and is allosterically regulated by the pathway end product, Leu. IPMS is a dimeric enzyme with each chain consisting of catalytic, accessory, and regulatory domains, with the accessory and regulatory domains of each chain sitting adjacent to the catalytic domain of the other chain. The IPMS crystal structure shows significant asymmetry because of different relative domain conformations in each chain.

Biosynthesis of Nodulisporic Acids: A Multifunctional Monooxygenase Delivers a Complex and Highly Branched Array.

Nodulisporic acids (NAs) are structurally complex potent antiinsectan indole diterpenes. We previously reported the biosynthetic gene cluster for these metabolites in Hypoxylon pulicicidum and functionally characterised the first five steps of the biosynthetic pathway. Here we reveal a highly complex biosynthetic array, furnishing multiple end products through expression of cluster components in Penicillium paxilli.

Precise Positioning of Water Is Critical for Hydrolysis Catalyzed by 5'-Methylthioadenosine Nucleosidase.

Enzyme-catalyzed hydrolysis is a fundamental chemical transformation involved in many essential metabolic processes. The enzyme 5'-methylthioadenosine/-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolysis of adenosine-containing metabolites in cysteine and methionine metabolism. Although MTAN enzymes contain highly similar active site architecture and generally follow a dissociative (D*A) reaction mechanism, substantial differences in reaction rates and chemical transition state structures have been reported.

Aminoacylation of Indole Diterpenes by Cluster-Specific Monomodular NRPS-like Enzymes.

Decoration of the core scaffolds of indole diterpene (IDT) natural products is key to generating structural and bioactivity diversity. Aminoacylation as a tailoring step is rarely linked to terpene biosynthesis and is extremely rare in IDT biosynthesis. Through heterologous pathway reconstruction, we have illuminated the genetic and biochemical basis for the only reported examples of aminoacylation in IDT biosynthesis, demonstrating the unusual involvement of monomodular nonribosomal peptide synthetase (NRPS)-like enzymes in IDT decoration.

Reciprocal allostery arising from a bienzyme assembly controls aromatic amino acid biosynthesis in Prevotella nigrescens.

Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bienzyme assembly comprising a 3-deoxy-d-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactions of aromatic amino acid biosynthesis in the bacterium Prevotella nigrescens (PniDAH7PS), respectively.

Diverse allosteric componentry and mechanisms control entry into aromatic metabolite biosynthesis.

Allosteric regulation of the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) controls the entry into aromatic amino acid biosynthesis in plants and microorganisms. DAH7PS has acquired a diverse range of allosteric machinery to enable this functionality. This review provides an overview of the current knowledge of the structural basis of allostery in this enzyme family and the evolutionary relationships between the different solutions to allosteric control of aromatic metabolite biosynthesis.

Medically actionable pathogenic variants in a population of 13,131 healthy elderly individuals.

Purpose: To measure the prevalence of medically actionable pathogenic variants (PVs) among a population of healthy elderly individuals.

Methods: We used targeted sequencing to detect pathogenic or likely pathogenic variants in 55 genes associated with autosomal dominant medically actionable conditions, among a population of 13,131 individuals aged 70 or older (mean age 75 years) enrolled in the ASPirin in Reducing Events in the Elderly (ASPREE) trial. Participants had no previous diagnosis or current symptoms of cardiovascular disease, physical disability or dementia, and no current diagnosis of life-threatening cancer.

A single amino acid substitution uncouples catalysis and allostery in an essential biosynthetic enzyme in .

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d--heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in , which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites.

Nodulisporic acid E biosynthesis: characterisation of NodD1, an indole-diterpene prenyltransferase that acts on an emindole SB derived indole-diterpene scaffold.

Prenylation of aromatic compounds is a key tailoring reaction in biosynthesis of bioactive indole-diterpenes. Here, we identify NodD1 as the enzyme responsible for the bisprenylation of nodulisporic acid F. This prenyltransferase showed a preference for its natural indole-diterpene substrate whereas other related enzymes were not able to catalyse this conversion.

Hinge Twists and Population Shifts Deliver Regulated Catalysis for ATP-PRT in Histidine Biosynthesis.

Allosteric regulation plays an important role in the control of metabolic flux in biosynthetic pathways. In microorganisms, many enzymes in these pathways adopt different strategies of allostery to allow the tuning of their activities in response to metabolic demand. Thus, it is important to uncover the mechanism of allosteric signal transmission to fully comprehend the complex control of enzyme function and its evolution.

Structural and functional characterisation of the entry point to pyocyanin biosynthesis in defines a new 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase subclass.

In (), the shikimate pathway end product, chorismate, serves as the last common precursor for the biosynthesis of both primary aromatic metabolites, including phenylalanine, tyrosine and tryptophan, and secondary aromatic metabolites, including phenazine-1-carboxylic acid (PCA) and pyocyanin (PYO). The enzyme 3-deoxy-d--heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, en route to chorismate. expresses multiple, distinct DAH7PSs that are associated with either primary or secondary aromatic compound biosynthesis.

'Tethering' fragment-based drug discovery to identify inhibitors of the essential respiratory membrane protein type II NADH dehydrogenase.

Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified.

Structure of the NDH-2 - HQNO inhibited complex provides molecular insight into quinone-binding site inhibitors.

Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO).

MIDAS: A Modular DNA Assembly System for Synthetic Biology.

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements.

A Pseudoisostructural Type II DAH7PS Enzyme from Pseudomonas aeruginosa: Alternative Evolutionary Strategies to Control Shikimate Pathway Flux.

The shikimate pathway is responsible for the biosynthesis of key aromatic metabolites in microorganisms and plants. The enzyme 3-deoxy-d- arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the pathway and DAH7PSs are classified as either type I or type II. The DAH7PSs from Pseudomonas aeruginosa are of particular interest as open reading frames encoding four putative DAH7PS isoenzymes, two classified as type Iα and two classified as type II, have been identified.

Exploring modular allostery via interchangeable regulatory domains.

Most proteins comprise two or more domains from a limited suite of protein families. These domains are often rearranged in various combinations through gene fusion events to evolve new protein functions, including the acquisition of protein allostery through the incorporation of regulatory domains. The enzyme 3-deoxy-d--heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of aromatic amino acid biosynthesis and displays a diverse range of allosteric mechanisms.

Draft Genome Sequence of the Filamentous Fungus ATCC 74245.

strain MF5954 (ATCC 74245) (formerly classified as sp.) is a filamentous fungal species known for its production of the secondary metabolite nodulisporic acid A. We present here the 41.