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Instruments / Technical Area	Publication Details	Published
I04-1-Macromolecular Crystallography (fixed wavelength)	Concentration‐dependent inhibition of mesophilic PETases on poly(ethylene terephthalate) can be eliminated by enzyme engineering 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 Chemsuschem DOI: 10.1002/cssc.202202277 Diamond Proposal Number(s): [17212] Show Abstract ▼ 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.	Feb 2023
I03-Macromolecular Crystallography	Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440 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 Metabolic Engineering 3 DOI: 10.1016/j.ymben.2023.02.006 Diamond Proposal Number(s): [23269] Open Access Show Abstract ▼ 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.	Feb 2023
B21-High Throughput SAXS I03-Macromolecular Crystallography I23-Long wavelength MX	Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation 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 Proceedings Of The National Academy Of Sciences 120 DOI: 10.1073/pnas.2212246120 Diamond Proposal Number(s): [23269] Open Access Show Abstract ▼ 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.	Jan 2023
I03-Macromolecular Crystallography	Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity 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 Nature Communications 13 DOI: 10.1038/s41467-022-35237-x Diamond Proposal Number(s): [23269] Open Access Show Abstract ▼ 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.	Dec 2022
I03-Macromolecular Crystallography	Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation 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 Chemsuschem DOI: 10.1002/cssc.202101932 Diamond Proposal Number(s): [23269] Show Abstract ▼ 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.	Sep 2021
B21-High Throughput SAXS	Using Small Angle X-Ray Scattering (SAXS) to characterize the solution conformation and flexibility of Matrix Metalloproteinases (MMPs) Louise E. Butt , Robert A. Holland , Nikul S. Khunti , Debra L. Quinn , Andrew R. Pickford DOI: 10.1007/978-1-4939-6863-3_6 Show Abstract ▼ Abstract: Small angle X-ray scattering (SAXS) provides information about the conformation and flexibility of proteins in solution, and hence provides complementary structural information to that obtained from X-ray crystallography and nuclear magnetic resonance spectroscopy. In this chapter, we describe the methods for the preparation of matrix metalloproteinase (MMP) samples for SAXS analyses, and for the acquisition, processing and interpretation of the SAXS data.	Mar 2017
I22-Small angle scattering & Diffraction	The interface between catalytic and hemopexin domains in matrix metalloproteinase-1 conceals a collagen binding exosite Laurence H. Arnold , Louise E. Butt , Stephen H. Prior , Christopher M. Read , Gregg B. Fields , Andrew R. Pickford Journal Of Biological Chemistry 286 (52), 45073-82 DOI: 10.1074/jbc.M111.285213 Diamond Proposal Number(s): [6419] Open Access Show Abstract ▼ Abstract: Matrix metalloproteinase-1 (MMP-1) is an instigator of collagenolysis, the catabolism of triple helical collagen. Previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilizing the collagen substrate in preparation for hydrolysis of the polypeptide backbone by the catalytic (CAT) domain. Here, we use biophysical methods to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen ?1(I) chain. The two components interact with 1:1 stoichiometry and micromolar affinity via a binding site within blades 1 and 2 of the four-bladed HPX domain propeller. Subsequent site-directed mutagenesis and assay implicates blade 1 residues Phe301, Val319, and Asp338 in collagen binding. Intriguingly, Phe301 is partially masked by the CAT domain in the crystal structure of full-length MMP-1 implying that transient separation of the domains is important in collagen recognition. However, mutation of this residue in the intact enzyme disrupts the CAT-HPX interface resulting in a drastic decrease in binding activity. Thus, a balanced equilibrium between these compact and dislocated states may be an essential feature of MMP-1 collagenase activity.	Nov 2011

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