ORCID Profile
0000-0001-8549-0122
Current Organisations
Chiang Mai University Faculty of Pharmacy
,
Chiang Mai University
,
Australian National University
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Publisher: Cold Spring Harbor Laboratory
Date: 15-02-2021
DOI: 10.1101/2021.02.14.431189
Abstract: Cyanobacteria have evolved a remarkably powerful CO 2 concentrating mechanism (CCM), enabling high photosynthetic rates in environments with limited inorganic carbon (Ci). Therefore, this CCM is a promising system for integration into higher plant chloroplasts to boost photosynthetic efficiency and yield. The CCM depends on active Ci uptake, facilitated by bicarbonate transporters and CO 2 pumps, to elevate CO 2 concentration around the active sites of the primary CO 2 fixing enzyme, Rubisco, which is encapsulated in cytoplasmic micro-compartments (carboxysomes). The essential CCM proteins have been identified, but the molecular signals and regulators that coordinate function in response to light, Ci availability and other environmental cues are largely unknown. Here, we provide evidence, based on a novel in vitro binding system, for a role of the PII-like SbtB protein in regulating Ci uptake by the bicarbonate transporter, SbtA, in response to the cellular adenylate energy charge (AEC) through dynamic protein-protein interaction. Binding of the SbtA and SbtB proteins from two phylogenetically distant species, Cyanobium sp . PCC7001 and Synechococcus elongatus PCC7942, was inhibited by high ATP, and promoted by low [ATP]:[ADP or AMP] ratios in vitro , consistent with a sensory response to the AEC mediated through adenylnucleotide ligand-specific conformation changes in SbtB. In vivo , cell cultures of S. elongatus showed up to 70% SbtB-dependent down-regulation of SbtA bicarbonate uptake activity specifically in the light activation phase during transitions from dark to low light when low cellular AEC is expected to limit metabolic activity. This suggests SbtB may function as a curfew protein during prolonged low cellular AEC and photosynthetically unfavourable conditions to prevent energetically futile and physiologically disadvantageous activation of SbtA.
Publisher: Springer Science and Business Media LLC
Date: 23-11-2020
DOI: 10.1038/S41467-020-19695-9
Abstract: Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including propargyl-DO3A-Gd(III) tagging and double electron–electron resonance (DEER) to study the rigid body protein dynamics of reconstructed evolutionary intermediates to determine how the conformational s ling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states, including a wide-open state, are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively s les catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3CC01578E
Abstract: Chemical stabilisation of carrier protein bound substrates in non-ribosomal peptide synthesis can result in a loss in activity of neighbouring catalytic domains.
Publisher: American Chemical Society (ACS)
Date: 11-08-2023
DOI: 10.1021/ACS.BIOCHEM.2C00323
Abstract: The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.
Publisher: eLife Sciences Publications, Ltd
Date: 06-2023
Abstract: Cyanobacteria have evolved a remarkably powerful CO2 concentrating mechanism (CCM), enabling high photosynthetic rates in environments with limited inorganic carbon (Ci). Therefore, this CCM is a promising system for integration into higher plant chloroplasts to boost photosynthetic efficiency and yield. The CCM depends on active Ci uptake, facilitated by bicarbonate transporters and CO2 pumps, to elevate CO2 concentration around the active sites of the primary CO2 fixing enzyme, Rubisco, which is encapsulated in cytoplasmic micro-compartments (carboxysomes). The essential CCM proteins have been identified, but the molecular signals and regulators that coordinate function in response to light, Ci availability and other environmental cues are largely unknown. Here, we provide evidence, based on a novel in vitro binding system, for a role of the PII-like SbtB protein in regulating Ci uptake by the bicarbonate transporter, SbtA, in response to the cellular adenylate energy charge (AEC) through dynamic protein-protein interaction. Binding of the SbtA and SbtB proteins from two phylogenetically distant species, Cyanobium sp. PCC7001 and Synechococcus elongatus PCC7942, was inhibited by high ATP, and promoted by low [ATP]:[ADP or AMP] ratios in vitro, consistent with a sensory response to the AEC mediated through adenylnucleotide ligand-specific conformation changes in SbtB. In vivo, cell cultures of S. elongatus showed up to 70% SbtB-dependent down-regulation of SbtA bicarbonate uptake activity specifically in the light activation phase during transitions from dark to low light when low cellular AEC is expected to limit metabolic activity. This suggests SbtB may function as a curfew protein during prolonged low cellular AEC and photosynthetically unfavourable conditions to prevent energetically futile and physiologically disadvantageous activation of SbtA.
Publisher: Frontiers Media SA
Date: 16-06-2023
Publisher: Springer Science and Business Media LLC
Date: 23-04-2018
DOI: 10.1038/S41589-018-0043-2
Abstract: The emergence of enzymes through the neofunctionalization of noncatalytic proteins is ultimately responsible for the extraordinary range of biological catalysts observed in nature. Although the evolution of some enzymes from binding proteins can be inferred by homology, we have a limited understanding of the nature of the biochemical and biophysical adaptations along these evolutionary trajectories and the sequence in which they occurred. Here we reconstructed and characterized evolutionary intermediate states linking an ancestral solute-binding protein to the extant enzyme cyclohexadienyl dehydratase. We show how the intrinsic reactivity of a desolvated general acid was harnessed by a series of mutations radiating from the active site, which optimized enzyme-substrate complementarity and transition-state stabilization and minimized s ling of noncatalytic conformations. Our work reveals the molecular evolutionary processes that underlie the emergence of enzymes de novo, which are notably mirrored by recent ex les of computational enzyme design and directed evolution.
Publisher: Cold Spring Harbor Laboratory
Date: 13-09-2022
DOI: 10.1101/2022.09.13.507756
Abstract: The emergence of oligomers is common during the evolution and ersification of protein families, yet the selective advantage of oligomerization is often cryptic or unclear. Oligomerization can involve the formation of isologous head-to-head interfaces (e.g., in symmetrical dimers) or heterologous head-to-tail interfaces (e.g., in cyclic complexes), the latter of which is less well studied and understood. In this work, we retrace the emergence of the trimeric form of cyclohexadienyl dehydratase from Pseudomonas aeruginosa (PaCDT) by introducing residues that form the PaCDT trimer-interface into AncCDT-5 (a monomeric reconstructed ancestor of PaCDT). We find that single interface mutations can switch the oligomeric state of the variants (implying evolutionarily metastable oligomeric states) and that trimerization corresponds with a reduction in the K M value of the enzyme from a promiscuous level to the physiologically relevant range. We show that this can be rationalized at the structural and dynamic level by reduced s ling of a non-catalytic conformational substate, and that trimerization was likely followed by a C-terminal extension that further refined the conformational s ling and kinetic properties of the enzyme. This work provides insight into how neutral s ling of metastable oligomeric states along an evolutionary trajectory can facilitate the evolution and optimization of enzyme function. Understanding how and why structural complexity (including homo-oligomerization and sequence insertions) emerges during the evolution and ersification of natural enzymes is a key goal in the study and design of protein function. We show that cyclic homo-oligomeric states can emerge via a small number of substitutions, and that trimerization and a C-terminal extension contributed to the tuning of catalytic properties during the evolution of cyclohexadienyl dehydratase from Pseudomonas aeruginosa .
Publisher: American Chemical Society (ACS)
Date: 20-11-2019
DOI: 10.1021/ACS.BIOCHEM.9B00880
Abstract: Cyanobacteria have evolved a suite of enzymes and inorganic carbon (C
Publisher: Elsevier BV
Date: 08-2019
DOI: 10.1016/J.SBI.2019.01.013
Abstract: Biosensors that selectively report on the presence of specific small molecule analytes have applications in many fields of research, medicine and biotechnology. Here, we review recent advances and emerging approaches in the design and optimisation of genetically encoded fluorescence-based small molecule biosensors. We discuss how natural sensory proteins can be exploited to produce novel biosensors and the strategies for optimizing ligand specificity and fluorescence readout. Finally, we provide insight into high-throughput sensor optimisation and discuss the challenges that are faced when designing novel biosensors.
Publisher: Elsevier BV
Date: 08-2023
Publisher: eLife Sciences Publications, Ltd
Date: 06-2023
DOI: 10.7554/ELIFE.88488
Abstract: Cyanobacteria have evolved a remarkably powerful CO2 concentrating mechanism (CCM), enabling high photosynthetic rates in environments with limited inorganic carbon (Ci). Therefore, this CCM is a promising system for integration into higher plant chloroplasts to boost photosynthetic efficiency and yield. The CCM depends on active Ci uptake, facilitated by bicarbonate transporters and CO2 pumps, to elevate CO2 concentration around the active sites of the primary CO2 fixing enzyme, Rubisco, which is encapsulated in cytoplasmic micro-compartments (carboxysomes). The essential CCM proteins have been identified, but the molecular signals and regulators that coordinate function in response to light, Ci availability and other environmental cues are largely unknown. Here, we provide evidence, based on a novel in vitro binding system, for a role of the PII-like SbtB protein in regulating Ci uptake by the bicarbonate transporter, SbtA, in response to the cellular adenylate energy charge (AEC) through dynamic protein-protein interaction. Binding of the SbtA and SbtB proteins from two phylogenetically distant species, Cyanobium sp. PCC7001 and Synechococcus elongatus PCC7942, was inhibited by high ATP, and promoted by low [ATP]:[ADP or AMP] ratios in vitro, consistent with a sensory response to the AEC mediated through adenylnucleotide ligand-specific conformation changes in SbtB. In vivo, cell cultures of S. elongatus showed up to 70% SbtB-dependent down-regulation of SbtA bicarbonate uptake activity specifically in the light activation phase during transitions from dark to low light when low cellular AEC is expected to limit metabolic activity. This suggests SbtB may function as a curfew protein during prolonged low cellular AEC and photosynthetically unfavourable conditions to prevent energetically futile and physiologically disadvantageous activation of SbtA.
Publisher: BMJ
Date: 06-2016
Publisher: Informa UK Limited
Date: 14-03-2014
DOI: 10.4161/CHAN.28136
Publisher: Wiley
Date: 28-11-2022
DOI: 10.1002/PRO.4510
Abstract: The emergence of oligomers is common during the evolution and ersification of protein families, yet the selective advantage of oligomerization is often cryptic or unclear. Oligomerization can involve the formation of isologous head‐to‐head interfaces (e.g., in symmetrical dimers) or heterologous head‐to‐tail interfaces (e.g., in cyclic complexes), the latter of which is less well studied and understood. In this work, we retrace the emergence of the trimeric form of cyclohexadienyl dehydratase from Pseudomonas aeruginosa (PaCDT) by introducing residues that form the PaCDT trimer‐interfaces into AncCDT‐5 (a monomeric reconstructed ancestor of PaCDT). We find that single interface mutations can switch the oligomeric state of the variants and that trimerization corresponds with a reduction in the K M value of the enzyme from a promiscuous level to the physiologically relevant range. In addition, we find that removal of a C‐terminal extension present in PaCDT leads to a variant with reduced catalytic activity, indicating that the C‐terminal region has a role in tuning enzymatic activity. We show that these observations can be rationalized at the structural and dynamic levels, with trimerization and C‐terminal extension leading to reduced s ling of non‐catalytic conformational substates in molecular dynamics simulations. Overall, this work provides insight into how neutral s ling of distinct oligomeric states along an evolutionary trajectory can facilitate the evolution and optimization of enzyme function.
Publisher: Cold Spring Harbor Laboratory
Date: 30-06-2017
DOI: 10.1101/157495
Abstract: Much of the functional ersity observed in modern enzyme superfamilies originates from molecular tinkering with existing enzymes 1 . New enzymes frequently evolve from enzymes with latent, promiscuous activities 2 , and often inherit key features of the ancestral enzyme, retaining conserved catalytic groups and stabilizing analogous intermediates or transition states 3 . While experimental evolutionary biochemistry has yielded considerable insight into the evolution of new enzymes from existing enzymes 4 , the emergence of catalytic activity de novo remains poorly understood. Although certain enzymes are thought to have evolved from non-catalytic proteins 5–7 , the mechanisms underlying these complete evolutionary transitions have not been described. Here we show how the enzyme cyclohexadienyl dehydratase (CDT) evolved from a cationic amino acid-binding protein belonging to the solute-binding protein (SBP) superfamily. Analysis of the evolutionary trajectory between reconstructed ancestors and extant proteins showed that the emergence and optimization of catalytic activity involved several distinct processes. The emergence of CDT activity was potentiated by the incorporation of a desolvated general acid into the ancestral binding site, which provided an intrinsically reactive catalytic motif, and reshaping of the ancestral binding site, which facilitated enzyme-substrate complementarity. Catalytic activity was subsequently gained via the introduction of hydrogen-bonding networks that positioned the catalytic residue precisely and contributed to transition state stabilization. Finally, catalytic activity was enhanced by remote substitutions that refined the active site structure and reduced s ling of non-catalytic states. Our work shows that the evolutionary processes that underlie the emergence of enzymes by natural selection in the wild are mirrored by recent ex les of computational design and directed evolution of enzymes in the laboratory.
Publisher: Cold Spring Harbor Laboratory
Date: 09-09-2019
DOI: 10.1101/762807
Abstract: Cyanobacteria have evolved a suite of enzymes and inorganic carbon (C i ) transporters that improve photosynthetic performance by increasing the localized concentration of CO 2 around the primary CO 2 -fixating enzyme, Rubisco. This CO 2 -concentrating mechanism (CCM) is highly regulated, responds to illumination/darkness cycles and allows cyanobacteria to thrive under limiting C i conditions. While the transcriptional control of CCM activity is well understood, less is known about how regulatory proteins might allosterically regulate C i transporters in response to changing conditions. Cyanobacterial sodium-dependent bicarbonate transporters (SbtAs) are inhibited by P II -like regulatory proteins (SbtBs), with the inhibitory effect being modulated by adenylnucleotides. Here, we used isothermal titration calorimetry to show that SbtB from Cyanobium sp. PCC7001 (SbtB7001) binds AMP, ADP, cAMP and ATP with micromolar-range affinities. X-ray crystal structures of apo- and nucleotide-bound SbtB7001 revealed that while AMP, ADP and cAMP have little effect on the SbtB7001 structure, binding of ATP stabilizes the otherwise flexible T-loop and that the flexible C-terminal C-loop adopts several distinct conformations. We also show that ATP binding affinity is increased ten-fold in the presence of Ca 2+ and we present an X-ray crystal structure of Ca 2+ ATP:SbtB7001 that shows how this metal ion facilitates additional stabilizing interactions with the apex of the T-loop. We propose that the Ca 2+ ATP-induced conformational change observed in SbtB7001 is important for allosteric regulation of SbtA activity by SbtB and is consistent with changing adenylnucleotide levels in illumination/darkness cycles.
Publisher: Cold Spring Harbor Laboratory
Date: 13-01-2020
DOI: 10.1101/2020.01.12.903682
Abstract: Antibodies targeting the NANP/NVDP repeat domain of the Plasmodium falciparum circumsporozoite protein (CSP Repeat ) can confer protection against malaria. However, it has also been suggested that this repeat domain exists as a decoy that distracts the immune system from mounting protective responses targeting other domains of CSP. Here we show that B cell responses to the repeat domain are indeed ∼10 fold higher than responses to the N- and C-terminal regions of CSP after sporozoite immunization. We investigated the role of the number of CSP Repeat -specific naïve precursor B cells and high avidity binding by B cells in driving the immunodominance of the CSP Repeat . Using adoptive transfer of germline precursors specific for the CSP Repeat , we found that increasing precursor number did indeed increase the responses to the repeat region, but not to the detriment of responses to other epitopes. To investigate the role of avid binding by B cells to the CSP Repeat in driving immunodominance we generated CSP9: a truncated CSP molecule with just 9 NANP repeats. Compared to near full length CSP molecules, CSP9 induced lower BCR signalling in CSP Repeat -specific cells and induced stronger responses to non-repeat epitopes. Finally, we found mice immunized with CSP9 molecules were strongly protected against mosquito bite challenge. Collectively these data demonstrate that the CSP Repeat does function as an immunodominant decoy and that truncated CSP molecules may be a promising avenue for future malaria vaccines. Malaria kills approximately 420,000 in iduals each year(1). Our best vaccine, RTS,S/AS01 is based on the circumsporozoite protein that coasts the surface of the parasite. However, this vaccine is only partially protective. Here we show that responses to a repeat region in the circumsporozoite dominate the immune response. However, immunizing with a circumsporozoite protein with a shortened repeat region induces a more erse immune response, which could be an avenue to make better malaria vaccines.
Publisher: Elsevier BV
Date: 09-2022
DOI: 10.1016/J.JMB.2022.167678
Abstract: Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera's activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen-deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.
Publisher: Springer Science and Business Media LLC
Date: 04-05-2021
DOI: 10.1038/S41467-021-22623-0
Abstract: Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
Publisher: American Chemical Society (ACS)
Date: 28-05-2021
Publisher: Public Library of Science (PLoS)
Date: 31-07-2017
Publisher: Elsevier BV
Date: 08-2021
Publisher: Elsevier BV
Date: 04-2021
Publisher: Research Square Platform LLC
Date: 06-01-2021
DOI: 10.21203/RS.3.RS-125509/V1
Abstract: Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report the first structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this previously uncharacterized complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.
Publisher: Cold Spring Harbor Laboratory
Date: 04-02-2020
DOI: 10.1101/2020.02.03.932491
Abstract: Several enzymes are known to have evolved from non-catalytic proteins such as solute-binding proteins (SBPs). Although attention has been focused on how a binding site can evolve to become catalytic, an equally important question is: how do the structural dynamics of a binding protein change as it becomes an efficient enzyme? Here we performed a variety of experiments, including double electron-electron resonance (DEER), on reconstructed evolutionary intermediates to determine how the conformational s ling of a protein changes along an evolutionary trajectory linking an arginine SBP to a cyclohexadienyl dehydratase (CDT). We observed that primitive dehydratases predominantly populate catalytically unproductive conformations that are vestiges of their ancestral SBP function. Non-productive conformational states are frozen out of the conformational landscape via remote mutations, eventually leading to extant CDT that exclusively s les catalytically relevant compact states. These results show that remote mutations can reshape the global conformational landscape of an enzyme as a mechanism for increasing catalytic activity.
Publisher: Proceedings of the National Academy of Sciences
Date: 31-01-2019
Abstract: The protein Ebony from Drosophila melanogaster plays a central role in the regulation of histamine and dopamine in various tissues through condensation of these amines with β-alanine. Ebony is a rare ex le of a nonribosomal peptide synthetase (NRPS) from a higher eukaryote and contains a C-terminal sequence that does not correspond to any previously characterized NRPS domain. We have structurally characterized this C-terminal domain and have discovered that it adopts the aryl-alkylamine- N -acetyl transferase (AANAT) fold, which is unprecedented in NRPS biology. Through analysis of ligand-bound structures, activity assays, and binding measurements, we have determined how this atypical condensation domain is able to provide selectivity for both the carrier protein-bound amino acid and the amine substrates, a situation that remains unclear for standard condensation domains identified to date from NRPS assembly lines. These results demonstrate that the C terminus of Ebony encodes a eukaryotic ex le of an alternative type of NRPS condensation domain they also illustrate how the catalytic components of such assembly lines are significantly more erse than a minimal set of conserved functional domains.
No related grants have been discovered for Joe Alexander Kaczmarski.