Emily Parker

Victoria University of Wellington · Ferrier Research Institute

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,...

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 gener...

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 determ...

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 an...

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 domai...

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 prod...

Nodulisporic acids are structurally complex potent antiinsectan natural products produced by the filamentous fungus Hypoxylon pulicicidum. A gene cluster, consisting of 13 genes, has been identified and biosynthetic machinery functionally characterised. This work reveals a highly complex biosynthetic array, furnishing multiple nodulisporic acid con...

Enzyme-catalyzed hydrolysis is a fundamental chemical transformation involved in many essential metabolic processes. The enzyme 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) catalyzes the hydrolysis of adenosine-containing metabolites in cysteine and methionine metabolism. Although MTAN enzymes contain highly similar active site...

Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactio...

Modular protein assembly has been widely reported as a mechanism for constructing allosteric machinery. Recently, a distinctive allosteric system has been identified in a bi-enzyme assembly comprising a 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS) and chorismate mutase (CM). These enzymes catalyze the first and branch point reactio...

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...

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles towards 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,...

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...

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 t...

Because of their special organization, multifunctional enzymes play crucial roles in improving the performance of metabolic pathways. For example, the bacterium Prevotella nigrescens contains a distinctive bifunctional protein comprising a 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAH7PS), catalyzing the first reaction of the biosynthet...

In Pseudomonas aeruginosa , 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 and pyocyanin. The enzyme 3-deoxy-D- arabino -heptuloso...

The side chain of D380 changes position (cyan to green) upon HQNO binding. The structures of wild-type NDH-2 (wheat) and the HQNO complex (white) are overlaid.

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. He...

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 ca...

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 interes...

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 str...

Significance Protein functions are often evolved from recombination of a limited suite of protein folds. Engineering desired function into proteins via manipulation of the genes, mimicking natural evolutionary processes, represents a promising approach to synthesizing useful molecular tools. Allosteric proteins play important roles in many biologic...

Hypoxylon pulicicidum strain MF5954 (ATCC 74245) (formerly classified as Nodulisporium sp.) is a filamentous fungal species known for its production of the secondary metabolite nodulisporic acid A. We present here the 41.5-Mb draft genome sequence for this organism.

Nodulisporic acids comprise a group of valuable indole diterpenes that exhibit potent insecticidal activities. We report the identification of a gene cluster in the genome of the filamentous fungus Hypoxylon pulicicidum (Nodulisporium sp.) that contains genes responsible for the biosynthesis of nodulisporic acids. Using Penicillium paxilli as a het...

ATP-phosphoribosyltransferase (ATP-PRT) catalyses the first committed step of histidine biosynthesis in plants and micro-organisms. Two forms of ATP-PRT have been reported, which differ in their molecular architecture and mechanism of allosteric regulation. The short-form ATP-PRT is a hetero-octamer, with four HisG chains that comprise only the cat...

Type II NADH:quinone oxidoreductase (NDH-2) is a respiratory enzyme found in the electron-transport chain of many species, with the exception of mammals. It is a 40–70 kDa single-subunit monotopic membrane protein that catalyses the oxidation of NADH and the reduction of quinone molecules via the cofactor FAD. NDH-2 is a promising new target for dr...

There are twenty-five published structures of Mycobacterium tuberculosis anthranilate phosphoribosyltransferase (Mtb-AnPRT) that use the same crystallization protocol. The structures include protein complexed with natural and alternative substrates, protein:inhibitor complexes, and variants with mutations of substrate-binding residues. Amongst thes...

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated target for antimicrobial drug design. The ATP-PRT enzyme catalyzes the reversible substitution reaction between phosphoribosyl pyrophosphate and ATP. The enzyme exists in two structural...

Phosphoribosyltransferases (PRTs) bind 5'-phospho-α-d-ribosyl-1'-pyrophosphate (PRPP) and transfer its phosphoribosyl group (PRib) to specific nucleophiles. Anthranilate PRT (AnPRT) is a promiscuous PRT that can phosphoribosylate both anthranilate and alternative substrates, and is the only example of a type III PRT. Comparison of the PRPP binding...

The first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), adopts a range of distinct allosteric regulation mechanisms in different organisms, related to different quaternary assemblies. DAH7PS from Mycobacterium tuberculosis (MtuDAH7PS) is a homotetramer, with the allosteric sites in close proximity t...

Protein backbone RMSD values during MD simulations. RMSD values of protein backbone atoms in MtuDAH7PSWT (green), MtuDAH7PSWTdimer (red) and MtuDAH7PSG232P (blue) are plotted against simulation time (ns). (TIFF)

Comparison between calculated RMSF values and temperature factors obtained from crystal structure. (A) Calculated chain-average RMSF values for MtuDAH7PSWT during MD simulation. (B) Square root values of the chain-average temperature factor obtained from ligand free crystal structure (PDB 3NV8) of MtuDAH7PSWT. (TIFF)

ITC data. ITC data obtained for (A) 30 μM MtuDAH7PSG232P with 3 mM Trp titrant; (B) 28 μM of MtuDAH7PSG232P with 900 μM Phe titrant; (C) 24 μM MtuDAH7PSG232P and a background of 50 μM Phe present in the cell with a 5 mM Trp titrant; (D) 32 μM MtuDAH7PSF227D with 5 mM Trp titrant; (E) 32 μM of MtuDAH7PSF227D with 600 μM Phe titrant and (F) 28 μM Mtu...

AUC data. Sedimentation velocity data for MtuDAH7PSG232P in the absence and presence of 100 μM Trp and 100 μM Phe. Data collected at 20°C and 50,000 rpm. (A), (B) and (C) are the data from MtuDAH7PSG232P collected at 0.09, 0.6 and 0.9 mg.mL-1 and (D), (E) and (F) are the data from MtuDAH7PSG232P collected in the presence of 100 μM Trp and 100 μM Ph...

Multifunctional proteins play a variety of roles in metabolism. Here we examine the catalytic function of the combined 3-deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) and chorismate mutase (CM) from Geobacillus sp. DAH7PS operates at the start of the biosynthetic pathway for aromatic metabolites, whereas CM operates in a dedicated bra...

ATP-phosphoribosyltransferase (ATP-PRT) catalyses the first step of histidine biosynthesis. Two different forms of ATP-PRT have been described; the homo-hexameric long form, and the hetero-octameric short form. Lactococcus lactis possesses the short form ATP-PRT comprising four subunits of HisGS , the catalytic subunit, and four subunits of HisZ, a...

Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyses the first committed step of the histidine biosynthesis in plants and microorganisms. Here we present the functional and structural characterisation of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisG...

Chirality plays a major role in recognition and interaction of biologically important molecules. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is the first enzyme of the shikimate pathway, which is responsible for the synthesis of aromatic amino acids in bacteria and plants, and a potential target for the development of a...

Significance Phenylketonuria and milder hyperphenylalaninemias constitute the most common inborn error of amino acid metabolism, usually caused by defective phenylalanine hydroxylase (PAH). Although a highly restricted diet prevents intellectual impairment during development, additional therapies are required to combat cognitive dysfunction, execut...

One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability and temperature-dependent regulation, for example. We have coined the phrase "macromolecular rate theory (MMRT...

Neisseria meningitidis 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (NmeDAH7PS) adopts a homotetrameric structure consisting of an extensive and a less extensive interface. Perturbation of the less extensive interface through a single mutation of a salt bridge (Arg126-Glu27) formed at the tetramer interface of all chains resulted in a dimer...

Differential scanning calorimetry showing thermal stability of NmeDAH7PSWT and NmeDAH7PSR126S. (PDF)

Interface analysis of NmeDAH7PSWT and NmeDAH7PSR126S. (PDF)

Sedimentation velocity data for NmeDAH7PSWT and NmeDAH7PSR126S. (PDF)

Allosteric regulation of protein function, the process by which binding of an effector molecule provokes a functional response from a distal site, is critical for metabolic pathways. Yet, the way the allosteric signal is communicated remains elusive, especially in dynamic, entropically-driven regulation mechanisms for which no major conformational...

Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg2+-dependent reaction between 5' phosphoribosyl-1'-pyrophosphate (PRPP) and anthranilate. The roles of residues predicted to be involved...

Allostery, where remote ligand binding alters protein function, is essential for the control of metabolism. Here, we have identified a highly sophisticated allosteric response that allows complex control of the pathway for aromatic amino acid biosynthesis in the pathogen Mycobacterium tuberculosis. This response is mediated by an enzyme complex for...

In this issue of Structure, Chen et al. present structures of the FERM-containing protein tyrosine phosphatase PTPN3 in complex with a phosphopeptide fragment of susbtrate epidermal growth factor receptor pathway substrate, providing detailed information on substrate specificity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Allosteric regulation of enzyme activity plays important metabolic roles. Here we review the allostery of enzymes of amino-acid metabolism conferred by a discrete domain known as the ACT domain. This domain of 60-70 residues has a βαββαβ topology leading to a four-stranded β4β1β3β2 antiparallel sheet with two antiparallel helices on one face. Exten...

N-Acetylneuraminic acid (NANA) is the most common naturally occurring sialic acid and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. NANA is synthesised in prokaryotes via a condensation reaction between phosphoenolpyruvate (PEP) and N-acetylmannosamine (ManNAc). This reaction is ca...

3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first committed step of the shikimate pathway, which produces the aromatic amino acids as well as many other aromatic metabolites. DAH7PS catalyses an aldol-like reaction between phosphoenolpyruvate and erythrose 4-phosphate. Three phosphoenolpyruvate mimics, (R)-phospholac...

Anthranilate phosphoribosyltransferase (AnPRT), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from 5'-phosphoribosyl 1'-pyrophosphate (PRPP) to anthranilate to form 5'-phosphoribosyl anthranilate (PRA). Mtb-AnPRT...

α-Isopropylmalate synthase (IPMS) catalyses the reaction between α-ketoisovalerate and acetyl coenzyme A (AcCoA) in the first step of leucine biosynthesis. IPMS is closely related to homocitrate synthase, which catalyses the reaction between AcCoA and the unbranched α-ketoacid α-ketoglutarate. Analysis of these enzymes suggests that several differe...

Many proteins adopt homomeric quaternary structures to support their biological function, including the first enzyme of the shikimate pathway that is ultimately responsible for the biosynthesis of the aromatic amino acids in plants and microorganisms. This enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), adopts a variety of di...

The increase in enzymatic rates with temperature up to an optimum temperature (Topt) is widely attributed to classical Arrhenius behaviour, and the decrease in enzymatic rates above Topt is ascribed to protein denaturation and/or aggregation. This account persists despite many investigators noting that denaturation is insufficient to explain the de...

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were...

One of the novel aspects of kiwifruit is the presence of high level of quinic acid which contributes to the flavour of the fruit. Quinic acid metabolism intersects with the shikimate pathway, which is responsible for the de novo biosynthesis of primary and secondary aromatic metabolites. The gene encoding the enzyme which catalyses the second step...

Neisseria meningitidis is the causative agent of meningitis and meningococcal septicaemia is a major cause of disease worldwide, resulting in brain damage and hearing loss, and can be fatal in a large proportion of cases. The enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first reaction in the shikimate pathway l...

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were...

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyses the first step of the shikimate pathway for the biosynthesis of aromatic amino acids. Allosteric regulation of Thermotoga maritima DAH7PS is mediated by L-Tyr binding to a discrete ACT regulatory domain appended to a core catalytic (β/α)8 barrel. Variants of T. maritima DAH7PS...

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the pathway to 3-deoxy-D-manno-octulosonate, a component in the cell wall of Gram-negative bacteria. KDO8PS is evolutionarily and structurally related to the first enzyme of the...

Tuberculosis (TB) is an infectious disease caused by the pathogen Mycobacterium tuberculosis (M. tuberculosis), killing about two million people worldwide each year. An increase in the prevalence of drug-resistant strains of M. tuberculosis in the past decades has renewed focus on the development of new drugs that can treat both drug-sensitive and...

The sialic acid N-acetylneuraminic acid (NANA) has a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. These pathogens coat themselves with polysialic acids, mimicking the exterior surface of mammalian cells and consequentially concealing the bacteria from the host's immune system. NANA is syn...

α-Isopropylmalate synthase (α-IPMS) is a multi-domain protein catalysing the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate. This reaction is the first committed step in the leucine biosynthetic pathway in bacteria and plants, and α-IPMS is allosterically regulated by this amino acid. Existing crys...

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase is an important biosynthetic enzyme in Mycobacterium tuberculosis and a potential drug target. We describe the synthesis, evaluation and docking of inhibitors with various chain lengths, which provide insight into the plasticity of the active site for inhibitor binding.

Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis (Mtb) AnPRT, al...

Allosteric regulation of protein function is a critical component of metabolic control. Its importance is underpinned by the diversity of mechanisms and its presence in all three domains of life. The first enzyme of the aromatic amino acid biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, shows remarkable variation in allosteric r...

Allostery is the process by which remote sites of a system are energetically coupled to elicit a functional response. The early models of allostery such as the Monod-Wyman-Changeux model and the Koshland-Némethy-Filmer model explain the allosteric behavior of multimeric proteins. However, these models do not explain how allostery arises from atomic...