9 research outputs found
Natural products: biosynthesis, antimicrobial properties and protein targets
The diversity of biosynthetic pathways in prokaryotes and eukaryotes has led to
numerous bioactive natural products (NPs) which occupy a vast chemical space. Despite
the current challenges in NP research, these molecules are still relevant today as they are
a major source of human medicine as well as being useful biological tools. The
elucidation of their biosynthetic pathways has also provided information about the
biochemical and biophysical properties of fascinating enzyme families such as the α-
oxoamine synthases (AOSs). The AOSs are an expanding group of pyridoxal 5’-
phosphate (PLP)-dependent enzymes, which are involved in the biosynthesis of several
important NP, including those essential for life. This study reports the characterization
and structural analysis of a unique AOS denoted as TamD from Pseudoalteromonas
tunicata. This enzyme plays a major role in tambjamine biosynthesis and consists of
both an acyl carrier protein (ACP) domain and a PLP-binding catalytic domain. UV/vis
spectroscopy and mass spectrometry (MS) of the recombinant TamD purified from E.
coli revealed that the enzyme forms a Schiff base with PLP via Lys380, which is
responsible for its characteristic yellow colour. It binds L-serine as a natural substrate
with a Kd of 5.01 ± 0.64 mM. This thesis also reports structural data for TamD from xray
crystallography at a resolution of 4.98 Å, which shows four molecules in the
asymmetric unit (ASU) suggesting the enzyme exist as a dimer. The absence of the Nterminal
region where the ACP domain is located in the crystal strucuture also suggests
intrinsic flexibility and disorder within that region.
With the increasing demand for new anti-infective therapies, investigations of the
molecular interactions between NPs and their protein targets are vital in understanding
the inhibition or activation properties of these molecules. The cysteine transpeptidases
known as sortases produced by Gram positive bacteria have been identified as attractive
targets for NP inhibitors. In this thesis, the molecular basis for the inhibition of
Streptococcus mutans sortase A (SrtA) by the plant flavonoid, trans-chalcone is
explored, using a combination of MS, enzyme kinetics, molecular modelling and x-ray
crystallography. This study reports the first high resolution crystal structure of the
H139A mutant of S. mutans SrtA, which reveals a unique N-terminal α-helix domain.
Trans-chalcone was found to inhibit the in vitro activity of S. mutans SrtA in a slow,
tight–binding manner, with a half maximal inhibitory concentration (IC50) of 5.0 ± 0.6
μM. The interaction resulted in a covalent adduct with the active site cysteine residue
(Cys205) via a Michael addition mechanism. Additionally, trans-chalcone showed
evidence of S. mutans anti-biofilm activity in a concentration dependent manner up to
250 μM with an efficacy cut-off point at higher concentations. These results indicate
that chalcone flavonoids are worth further investigation as potential antibiofilm
inhibitors.
A renewed interest in plant NPs has also led to a collaborative investigation on the
antimicrobial potential of garlic-derived allicin, against Burkholderia cepacia complex
(Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant
and life-threatening human pathogen. Allicin is the principal antibacterial agent in fresh
preparations of garlic extracts. This investigation reports the first evidence that allicin
and allicin-contaning garlic extracts possess inhibitory and bactericidal activities against
Bcc. The minimum inhibitory concentrations (MICs) of aqueous garlic extract (AGE)
against 38 Bcc isolates ranged from 0.5 to 3% (v/v). An investigation into the possible
molecular mechanisms of allicin with a recombinant thiol-dependent peroxiredoxin
(BCP) from B. cenocepacia revealed that allicin and AGE modify an essential BCP
catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent
that targets protein thiols. Present therapeutic options against these life-threatening
pathogens are limited; thus, allicin-containing compounds merit further investigation as
adjuncts to existing antibiotics
A small-molecule activator of kinesin-1 drives remodeling of the microtubule network
The microtubule motor kinesin-1 interacts via its cargo-binding domain with both microtubules and organelles, and hence plays an important role in controlling organelle transport and microtubule dynamics. In the absence of cargo, kinesin-1 is found in an autoinhibited conformation. The molecular basis of how cargo engagement affects the balance between kinesin-1's active and inactive conformations and roles in microtubule dynamics and organelle transport is not well understood. Here we describe the discovery of kinesore, a small molecule that in vitro inhibits kinesin-1 interactions with short linear peptide motifs found in organelle-specific cargo adaptors, yet activates kinesin-1's function of controlling microtubule dynamics in cells, demonstrating that these functions are mechanistically coupled. We establish a proof-of-concept that a microtubule motor-cargo interface and associated autoregulatory mechanism can be manipulated using a small molecule, and define a target for the modulation of microtubule dynamics
Molecular basis of Streptococcus mutans sortase A inhibition by the flavonoid natural product trans-chalcone
Garlic Revisited: Antimicrobial Activity of Allicin-Containing Garlic Extracts against Burkholderia cepacia Complex
The antimicrobial activities of garlic and other plant alliums are primarily based on allicin, a thiosulphinate present in crushed garlic bulbs. We set out to determine if pure allicin and aqueous garlic extracts (AGE) exhibit antimicrobial properties against the Burkholderia cepacia complex (Bcc), the major bacterial phytopathogen for alliums and an intrinsically multiresistant and life-threatening human pathogen. We prepared an AGE from commercial garlic bulbs and used HPLC to quantify the amount of allicin therein using an aqueous allicin standard (AAS). Initially we determined the minimum inhibitory concentrations (MICs) of the AGE against 38 Bcc isolates; these MICs ranged from 0.5 to 3% (v/v). The antimicrobial activity of pure allicin (AAS) was confirmed by MIC and minimum bactericidal concentration (MBC) assays against a smaller panel of five Bcc isolates; these included three representative strains of the most clinically important species, B. cenocepacia. Time kill assays, in the presence of ten times MIC, showed that the bactericidal activity of AGE and AAS against B. cenocepacia C6433 correlated with the concentration of allicin. We also used protein mass spectrometry analysis to begin to investigate the possible molecular mechanisms of allicin with a recombinant form of a thiol-dependent peroxiredoxin (BCP, Prx) from B. cenocepacia. This revealed that AAS and AGE modifies an essential BCP catalytic cysteine residue and suggests a role for allicin as a general electrophilic reagent that targets protein thiols. To our knowledge, we report the first evidence that allicin and allicin-containing garlic extracts possess inhibitory and bactericidal activities against the Bcc. Present therapeutic options against these life-threatening pathogens are limited; thus, allicin-containing compounds merit investigation as adjuncts to existing antibiotics
X-ray diffraction data for Streptococcus mutans SrtAN40
3600 oscillation images of single crystal collected using a Pilatus 6M detector at Diamond Light Source Beamline I04-1. Images in compressed bzip archive.Molecular Basis of Streptococcus mutans Sortase A Inhibition by Chalcone. X-ray diffraction data from a single crystal of S. mutans SrtA-N40 truncation. Crystal produced from the following conditions: 1ul drops of protein at 10 mg/ml plus 1ul of well solution containing: 30 % w/v peg4000, 0.1 M Sodium acetate pH 4.6, 0.2 M Ammonium sulfate. 1 ml final volume in well. Data collected at Diamond Light Source 8th March 2014. Beam line I04-1. Structural model deposited in wwPDB as 4TQX. ## Note ## The file should be de-compressed with the bz2 algorithm and then untarred e.g. using 7-zip on Windows or "tar -tf" on Linux.Marles-Wright, Jon; Campopiano, Dominic; Wallock-Richards, Daynea. (2016). X-ray diffraction data for Streptococcus mutans SrtAN40, [dataset]. University of Edinburgh. http://dx.doi.org/10.7488/ds/1308
MICs (µg/ml) of AGE for <i>B. cepacia</i> complex.
<p>* <b>We have included all codes to avoid confusion between Edinburgh strain collection numbers, LMG and ATCC codes.</b></p><p>MICs (µg/ml) of AGE for <i>B. cepacia</i> complex.</p
MICs and MBCs (µg/ml) of AAS for <i>B. cepacia</i> complex.
<p>MICs and MBCs (µg/ml) of AAS for <i>B. cepacia</i> complex.</p
Chemical structure of allicin and mechanism of formation from alliin by the enzyme alliinase.
<p>Step 1. Alliinase hydrolyses alliin to produce allylsulfenic acid which, in step 2, condenses spontaneously with the loss of water to produce allicin.</p
HPLC and MS analysis of aqueous allicin standard (AAS) and aqueous garlic extract (AGE).
<p>(A) AAS was analysed using a C18 reverse phase column with UV detection at 240 nm. The standard eluted at 4.1 minutes. AAS had an observed <i>m/z</i> of 185.0067 (consistent with the [M+Na]<sup>+</sup> species; theoretical <i>m/z</i> 185.0065; [C<sub>6</sub>H<sub>10</sub>OS<sub>2</sub>+Na]<sup>+</sup>) (<i>inset</i>). (B) AGE was analysed by the same method as described for AAS. The peak at 4.1 minutes corresponds to the mass of allicin.</p