I03-Macromolecular Crystallography
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Alissa
Bleem
,
Eugene
Kuatsjah
,
Gerald N.
Presley
,
Daniel J.
Hinchen
,
Michael
Zahn
,
David C.
Garcia
,
William E.
Michener
,
Gerhard
König
,
Konstantinos
Tornesakis
,
Marco N.
Allemann
,
Richard J.
Giannone
,
John E.
Mcgeehan
,
Gregg T.
Beckham
,
Joshua K.
Michener
Abstract: Aryl-O-demethylation is a common rate-limiting step in the catabolism of lignin-related compounds, including guaiacol. Here we used randomly barcoded transposon insertion sequencing (RB-TnSeq) in the bacterium Novosphingobium aromaticivorans to identify a Rieske-type guaiacol O-demethylase, GdmA. Similarity searches identified GdmA homologs in other bacteria, along with candidate reductase partners, denoted GdmB. GdmAB combinations were biochemically characterized for activity with several lignin-related substrates. Structural and sequence comparisons of vanillate- and guaiacol-specific O-demethylase active sites revealed conserved hallmarks of substrate specificity. GdmAB combinations were also evaluated in Pseudomonas putida KT2440, which does not natively utilize guaiacol. GdmAB from Cupriavidus necator N-1 demonstrated the highest rate of guaiacol turnover in vitro and in engineered P. putida strains and notably higher catalytic efficiency than a cytochrome P450 system (GcoAB) and the vanillate Rieske-type O-demethylase from P. putida (VanAB). The GdmAB O-demethylases described here expand the suite of options for microbial conversion of a model lignin-derived substrate.
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May 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Several bacteria possess components of catabolic pathways for the synthetic polyester poly(ethylene terephthalate) (PET). These proceed by hydrolyzing the ester linkages of the polymer to its monomers, ethylene glycol and terephthalate (TPA), which are further converted into common metabolites. These pathways are crucial for genetically engineering microbes for PET upcycling, prompting interest in their fundamental biochemical and structural elucidation. Terephthalate dioxygenase (TPADO) and its cognate reductase make up a complex multimetalloenzyme system that dihydroxylates TPA, activating it for enzymatic decarboxylation to yield protocatechuic acid (PCA). Here, we report structural, biochemical, and bioinformatic analyses of TPADO. Together, these data illustrate the remarkable adaptation of TPADO to the TPA dianion as its preferred substrate, with small, protonatable ring 2-carbon substituents being among the few permitted substrate modifications. TPADO is a Rieske [2Fe2S] and mononuclear nonheme iron-dependent oxygenase (Rieske oxygenase) that shares low sequence similarity with most structurally characterized members of its family. Structural data show an α-helix–associated histidine side chain that rotates into an Fe (II)–coordinating position following binding of the substrate into an adjacent pocket. TPA interactions with side chains in this pocket were not conserved in homologs with different substrate preferences. The binding mode of the less symmetric 2-hydroxy-TPA substrate, the observation that PCA is its oxygenation product, and the close relationship of the TPADO α-subunit to that of anthranilate dioxygenase allowed us to propose a structure-based model for product formation. Future efforts to identify, evolve, or engineer TPADO variants with desirable properties will be enabled by the results described here.
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Mar 2022
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I03-Macromolecular Crystallography
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Eugene
Kuatsjah
,
Christopher W.
Johnson
,
Davinia
Salvachúa
,
Allison Z.
Werner
,
Michael
Zahn
,
Caralyn J.
Szostkiewicz
,
Christine A.
Singer
,
Graham
Dominick
,
Ikenna
Okekeogbu
,
Stefan J.
Haugen
,
Sean P.
Woodworth
,
Kelsey J.
Ramirez
,
Richard J.
Giannone
,
Robert L.
Hettich
,
John E.
Mcgeehan
,
Gregg T.
Beckham
Diamond Proposal Number(s):
[23269]
Abstract: The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Overall, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.
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Jan 2022
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I03-Macromolecular Crystallography
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Erika
Erickson
,
Thomas J.
Shakespeare
,
Felicia
Bratti
,
Bonnie L.
Buss
,
Rosie
Graham
,
Mckenzie A.
Hawkins
,
Gerhard
König
,
William E.
Michener
,
Joel
Miscall
,
Kelsey J.
Ramirez
,
Nicholas A.
Rorrer
,
Michael
Zahn
,
Andrew R.
Pickford
,
John E.
Mcgeehan
,
Gregg
Beckham
Diamond Proposal Number(s):
[23269]
Abstract: There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, we compare the Ideonella sakaiensis PETase wild-type enzyme to a previously reported improved variant (W159H/S238F). We compare the thermostability of each enzyme and describe a 1.45 Å resolution structure of the mutant, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies show that reaction temperature has the strongest influence on performance between the two enzymes. We also show that both enzymes achieve higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, we assess the impact of product accumulation on reaction progress for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displays different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both are sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme-
based polyester recycling.
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Sep 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212]
Abstract: An estimated 11 million tons of plastic waste enter our oceans annually, impacting wildlife, our food chain and our health. The UK government has set ambitious 2025 targets for plastics recycling, but effective means of achieving these are currently lacking. Therefore, an international team of researchers is focusing on the discovery and engineering of enzymes that can help break
down plastics for recycling.
In previous work, they characterised the structure and function of PETase, a bacterial enzyme with the remarkable ability to deconstruct one of the most commonly polluting thermoplastics, polyethylene terephthalate, PET. This study looked in-depth at a partner enzyme called MHETase, secreted from the same bacterium, that can significantly speed up the breakdown process.
The structures they collected on Diamond Light Source’s I03 Macromolecular Crystallography (MX) beamline are the highest resolution available and provide a detailed insight into the MHETase enzyme. Combined with detailed bioinformatics, biochemistry and molecular simulations, they show a highly synergistic relationship between the PETase and MHETase enzymes. The team investigated if tethering the proteins together could improve the breakdown and demonstrated that this was significantly faster than PETase alone or a PETase-MHETase cocktail.
Enzymes offer a low-energy solution and the potential to allow infinite recycling, reducing our growing requirements for fossil resources. There is a lot of excitement around the potential for naturally-evolved enzymes to tackle our plastic waste. This publication reached the Altmetric Top 100 (#39) from the 3.4 million papers published in 2020.
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Jul 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Emerald S.
Ellis
,
Daniel J.
Hinchen
,
Alissa
Bleem
,
Lintao
Bu
,
Sam J. B.
Mallinson
,
Mark D.
Allen
,
Bennett R.
Streit
,
Melodie M.
Machovina
,
Quinlan V.
Doolin
,
William E.
Michener
,
Christopher W.
Johnson
,
Brandon C.
Knott
,
Gregg T.
Beckham
,
John E.
Mcgeehan
,
Jennifer L.
Dubois
Diamond Proposal Number(s):
[17212, 23269]
Open Access
Abstract: Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.
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Feb 2021
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I03-Macromolecular Crystallography
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Brandon C.
Knott
,
Erika
Erickson
,
Mark D.
Allen
,
Japheth E.
Gado
,
Rosie
Graham
,
Fiona L.
Kearns
,
Isabel
Pardo
,
Ece
Topuzlu
,
Jared
Anderson
,
Harry P.
Austin
,
Graham
Dominick
,
Christopher W.
Johnson
,
Nicholas A.
Rorrer
,
Caralyn J.
Szostkiewicz
,
Valérie
Copié
,
Christina M.
Payne
,
H. Lee
Woodcock
,
Bryon S.
Donohoe
,
Gregg T.
Beckham
,
John E.
Mcgeehan
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.
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Sep 2020
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I04-Macromolecular Crystallography
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Melodie M.
Machovina
,
Sam J. B.
Mallinson
,
Brandon C.
Knott
,
Alexander W.
Meyers
,
Marc
Garcia-Borràs
,
Lintao
Bu
,
Japheth E.
Gado
,
April
Oliver
,
Graham P.
Schmidt
,
Daniel J.
Hinchen
,
Michael F.
Crowley
,
Christopher W.
Johnson
,
Ellen L.
Neidle
,
Christina M.
Payne
,
Kendall N.
Houk
,
Gregg T.
Beckham
,
John E.
Mcgeehan
,
Jennifer L.
Dubois
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.
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Jun 2019
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[14891]
Open Access
Abstract: Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-β amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset.
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Apr 2019
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Sam J. B.
Mallinson
,
Melodie M.
Machovina
,
Rodrigo L.
Silveira
,
Marc
Garcia-Borràs
,
Nathan
Gallup
,
Christopher W.
Johnson
,
Mark D.
Allen
,
Munir S.
Skaf
,
Michael F.
Crowley
,
Ellen L.
Neidle
,
Kendall N.
Houk
,
Gregg T.
Beckham
,
Jennifer L.
Dubois
,
John E.
Mcgeehan
Open Access
Abstract: Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion.
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Jun 2018
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