I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Cytochromes P450 (P450s) are a superfamily of heme-containing enzymes possessing a broad range of monooxygenase activities. One such activity is O-demethylation, an essential and rate-determining step in emerging strategies to valorize lignin that employ carbon-carbon bond cleavage. We recently identified PbdA, a P450 from Rhodococcus jostii RHA1, and PbdB, its cognate reductase, which catalyze the O-demethylation of para-methoxylated benzoates (p-MBAs) to initiate growth of RHA1 on these compounds. PbdA had the highest affinity (Kd = 3.8 ± 0.6 μM) and apparent specificity (kcat/KM = 20 000 ± 3 000 M-1 s-1) for p-MBA. The enzyme also O-demethylated two related lignin-derived aromatic compounds with remarkable efficiency: veratrate and isovanillate. PbdA also catalyzed the hydroxylation and dehydrogenation of p-EB even though RHA1 did not grow on this compound. Atomic-resolution structures of PbdA in complex with p-MBA, p-EB and veratrate revealed a cluster of three residues that form hydrogen bonds with the substrates’ carboxylate: Ser87, Ser237 and Arg84. Substitution of these residues resulted in lower affinity and O-demethylation activity on p-MBA as well as increased affinity for the acetyl analogue, p-methoxyacetophenone. The S87A and S237A variants of PbdA also catalyzed the O-demethylation of an aldehyde analogue of p-MBA, p-methoxy-benzaldehyde, while the R84M variant did not, despite binding this compound with high affinity. These results suggest that Ser87, Ser237 and Arg84 are not only important determinants of specificity but also help to orientate that substrate correctly in the active site. This study facilitates the design of biocatalysts for lignin valorization.
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Sep 2024
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I03-Macromolecular Crystallography
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Open Access
Abstract: Molecules that induce novel interactions between proteins hold great promise for the study of biological systems and the development of therapeutics, but their discovery has been limited by the complexities of rationally designing interactions between three components, and because known binders to each protein are typically required to inform initial designs. Here, we report a general and rapid method for discovering α-helically constrained (Helicon) polypeptides that cooperatively induce the interaction between two target proteins without relying on previously known binders or an intrinsic affinity between the proteins. We show that Helicons are capable of binding every major class of E3 ubiquitin ligases, which are of great biological and therapeutic interest but remain largely intractable to targeting by small molecules. We then describe a phage-based screening method for discovering “trimerizer” Helicons, and apply it to reprogram E3s to cooperatively bind an enzyme (PPIA), a transcription factor (TEAD4), and a transcriptional coactivator (β-catenin).
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Nov 2023
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I03-Macromolecular Crystallography
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Kevin J.
Mcnaught
,
Eugene
Kuatsjah
,
Michael
Zahn
,
Érica T.
Prates
,
Huiling
Shao
,
Gayle J.
Bentley
,
Andrew R.
Pickford
,
Josephine N.
Gruber
,
Kelley V.
Hestmark
,
Daniel A.
Jacobson
,
Brenton C.
Poirier
,
Chen
Ling
,
Myrsini
San Marchi
,
William E.
Michener
,
Carrie D.
Nicora
,
Jacob N.
Sanders
,
Caralyn J.
Szostkiewicz
,
Dušan
Veličković
,
Mowei
Zhou
,
Nathalie
Munoz
,
Young-Mo
Kim
,
Jon K.
Magnuson
,
Kristin E.
Burnum-Johnson
,
Kendall N.
Houk
,
John E.
Mcgeehan
,
Christopher W.
Johnson
,
Gregg T.
Beckham
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional β-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modelling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.
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Feb 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Luisana
Avilan
,
Bruce R.
Lichtenstein
,
Gerhard
Koenig
,
Michael
Zahn
,
Mark D.
Allen
,
Liliana
Oliveira
,
Matilda
Clark
,
Victoria
Bemmer
,
Rosie
Graham
,
Harry P.
Austin
,
Graham
Dominick
,
Christopher W.
Johnson
,
Gregg T.
Beckham
,
John
Mcgeehan
,
Andrew R.
Pickford
Diamond Proposal Number(s):
[17212]
Open Access
Abstract: Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. Here, we report that this inhibition is dependent on incubation time, the solution conditions and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which our simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis, and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.
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Feb 2023
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
I23-Long wavelength MX
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Eugene
Kuatsjah
,
Michael
Zahn
,
Xiangyang
Chen
,
Ryo
Kato
,
Daniel J.
Hinchen
,
Mikhail O.
Konev
,
Rui
Katahira
,
Christian
Orr
,
Armin
Wagner
,
Yike
Zou
,
Stefan J.
Haugen
,
Kelsey J.
Ramirez
,
Joshua K.
Michener
,
Andrew R.
Pickford
,
Naofumi
Kamimura
,
Eiji
Masai
,
Kendall N.
Houk
,
John
Mcgeehan
,
Gregg T.
Beckham
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon–carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.
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Jan 2023
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I03-Macromolecular Crystallography
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Erika
Erickson
,
Japheth E.
Gado
,
Luisana
Avilán
,
Felicia
Bratti
,
Richard K.
Brizendine
,
Paul A.
Cox
,
Raj
Gill
,
Rosie
Graham
,
Dong-Jin
Kim
,
Gerhard
König
,
William E.
Michener
,
Saroj
Poudel
,
Kelsey J.
Ramirez
,
Thomas J.
Shakespeare
,
Michael
Zahn
,
Eric S.
Boyd
,
Christina M.
Payne
,
Jennifer L.
Dubois
,
Andrew R.
Pickford
,
Gregg T.
Beckham
,
John E.
Mcgeehan
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
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Dec 2022
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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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