19 research outputs found
Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro
This work was funded by The Novo Nordisk Foundation grant to the Center for Biosustainability (NNF10CC1016517). P.I.N. was funded by grants from The Novo Nordisk Foundation (NNF20CC0035580, and LiFe, NNF18OC0034818), the European Unionâs Horizon 2020 Research and Innovation Programme under grant agreement No. 814418 (SinFonia) and the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B). T.K. and M.N.D. were funded by fellowships from the European Union's Horizon 2020 research and innovation program under a Marie SkĆodowska Curie project under grant agreement No. 713683 (COFUNDfellowsDTU).The fluorinase enzyme represents the only biological mechanism capable of forming stable CâF bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding â likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.Publisher PDFPeer reviewe
Reprogramming potential of the NRPS machinery related to biosynthesis of glycopeptide antibiotic teicoplanin
Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics
Redesign of substrate selection in glycopeptide antibiotic biosynthesis enables effective formation of alternate peptide backbones
Non-ribosomal peptide synthesis is capable of utilizing a wide range of amino acid residues due the selectivity of adenylation (A)-domains. Changing the selectivity of A-domains could lead to new bioactive non-ribosomal peptides, although remodeling efforts of A-domains are often unsuccessful. Here, we explored and successfully reengineered the specificity of the module 3 A-domain from glycopeptide antibiotic biosynthesis to change the incorporation of 3,5-dihydroxyphenylglycine into 4-hydroxyphenylglycine. These engineered A-domains remain selective in a functioning peptide assembly line even under substrate competition conditions, and indicating a possible application of these for the future redesign of GPA biosynthesis
The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis
Bacterial toxinâantitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxinâantitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_É1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_É1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence
Understanding the early stages of peptide formation during the biosynthesis of teicoplanin and related glycopeptide antibiotics
The biosynthesis of the glycopeptide antibiotics (GPAs) demonstrates the exceptional ability of nonâribosomal peptide synthesis to generate diverse and complex structures from an expanded array of amino acid precursors. Whilst the heptapeptide cores of GPAs share a conserved Câterminus, including the aromatic residues involved crosslinking and that are essential for the antibiotic activity of GPAs, most structural diversity is found within the Nâterminus of the peptide. Furthermore, the origin of the (D)âstereochemistry of residue 1 of all GPAs is currently unclear, despite its importance for antibiotic activity. Given these important features, we have now reconstituted modules 1â4 of the nonâribosomal peptide synthetase (NRPS) assembly lines that synthesise the clinicallyârelevant type IV GPA teicoplanin and the related compound A40926. Our results show that important roles in amino acid modification during the NRPSâmediated biosynthesis of GPAs can be ascribed to the actions of condensation domains present within these modules, including the incorporation of (D)âamino acids at position 1 of the peptide. Our results also indicate that hybrid NRPS assembly lines can be generated in a facile manner by mixing NRPS proteins from different systems, and that uncoupling of peptide formation due to different rates of activity seen for NRPS modules can be controlled by varying the ratio of NRPS modules. Taken together, this indicates that NRPS assembly lines function as dynamic peptide assembly lines and not static megaenzyme complexes, which has significant implications for biosynthetic redesign of these important biosynthetic systems
Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites <i>in vitro</i>
The fluorinase enzyme represents the only biological mechanism capable of forming stable CâF bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding â likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system
The seismo-hydromechanical behavior during deep geothermal reservoir stimulations: open questions tackled in a decameter-scale in situ stimulation experiment
In this
contribution, we present a review of scientific research results that address
seismo-hydromechanically coupled processes relevant for the development of a
sustainable heat exchanger in low-permeability crystalline rock and introduce
the design of the In situ Stimulation and Circulation (ISC) experiment at the
Grimsel Test Site dedicated to studying such processes under controlled
conditions. The review shows that research on reservoir stimulation for deep
geothermal energy exploitation has been largely based on laboratory
observations, large-scale projects and numerical models. Observations of
full-scale reservoir stimulations have yielded important results. However,
the limited access to the reservoir and limitations in the control on the
experimental conditions during deep reservoir stimulations is insufficient to
resolve the details of the hydromechanical processes that would enhance
process understanding in a way that aids future stimulation design.
Small-scale laboratory experiments provide fundamental insights into various
processes relevant for enhanced geothermal energy, but suffer from
(1)Â difficulties and uncertainties in upscaling the results to the field
scale and (2)Â relatively homogeneous material and stress conditions that lead
to an oversimplistic fracture flow and/or hydraulic fracture propagation
behavior that is not representative of a heterogeneous reservoir. Thus, there
is a need for intermediate-scale hydraulic stimulation experiments with high
experimental control that bridge the various scales and for which access to
the target rock mass with a comprehensive monitoring system is possible. The
ISC experiment is designed to address open research questions in a naturally
fractured and faulted crystalline rock mass at the Grimsel Test Site
(Switzerland). Two hydraulic injection phases were executed to enhance the
permeability of the rock mass. During the injection phases the rock mass
deformation across fractures and within intact rock, the pore pressure
distribution and propagation, and the microseismic response were monitored at
a high spatial and temporal resolution
The seismo-hydromechanical behavior during deep geothermal reservoir stimulations: open questions tackled in a decameter-scale in situ stimulation experiment
In this
contribution, we present a review of scientific research results that address
seismo-hydromechanically coupled processes relevant for the development of a
sustainable heat exchanger in low-permeability crystalline rock and introduce
the design of the In situ Stimulation and Circulation (ISC) experiment at the
Grimsel Test Site dedicated to studying such processes under controlled
conditions. The review shows that research on reservoir stimulation for deep
geothermal energy exploitation has been largely based on laboratory
observations, large-scale projects and numerical models. Observations of
full-scale reservoir stimulations have yielded important results. However,
the limited access to the reservoir and limitations in the control on the
experimental conditions during deep reservoir stimulations is insufficient to
resolve the details of the hydromechanical processes that would enhance
process understanding in a way that aids future stimulation design.
Small-scale laboratory experiments provide fundamental insights into various
processes relevant for enhanced geothermal energy, but suffer from
(1)Â difficulties and uncertainties in upscaling the results to the field
scale and (2)Â relatively homogeneous material and stress conditions that lead
to an oversimplistic fracture flow and/or hydraulic fracture propagation
behavior that is not representative of a heterogeneous reservoir. Thus, there
is a need for intermediate-scale hydraulic stimulation experiments with high
experimental control that bridge the various scales and for which access to
the target rock mass with a comprehensive monitoring system is possible. The
ISC experiment is designed to address open research questions in a naturally
fractured and faulted crystalline rock mass at the Grimsel Test Site
(Switzerland). Two hydraulic injection phases were executed to enhance the
permeability of the rock mass. During the injection phases the rock mass
deformation across fractures and within intact rock, the pore pressure
distribution and propagation, and the microseismic response were monitored at
a high spatial and temporal resolution