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1.
Biochemistry ; 2022 May 25.
Article in English | MEDLINE | ID: mdl-35612958

ABSTRACT

Enhancing the thermostability of enzymes without impacting their catalytic function represents an important yet challenging goal in protein engineering and biocatalysis. We recently introduced a novel method for enzyme thermostabilization that relies on the computationally guided installation of genetically encoded thioether "staples" into a protein via cysteine alkylation with the noncanonical amino acid O-2-bromoethyl tyrosine (O2beY). Here, we demonstrate the functionality of an expanded set of electrophilic amino acids featuring chloroacetamido, acrylamido, and vinylsulfonamido side-chain groups for protein stapling using this strategy. Using a myoglobin-based cyclopropanase as a model enzyme, our studies show that covalent stapling with p-chloroacetamido-phenylalanine (pCaaF) provides higher stapling efficiency and enhanced stability (thermodynamic and kinetic) compared to the other stapled variants and the parent protein. Interestingly, molecular simulations of conformational flexibility of the cross-links show that the pCaaF staple allows fewer energetically feasible conformers than the other staples, and this property may be a broader indicator of stability enhancement. Using this strategy, pCaaF-stapled variants with significantly enhanced stability against thermal denaturation (ΔTm' = +27 °C) and temperature-induced heme loss (ΔT50 = +30 °C) were obtained while maintaining high levels of catalytic activity and stereoselectivity. Crystallographic analyses of singly and doubly stapled variants provide key insights into the structural basis for stabilization, which includes both direct interactions of the staples with protein residues and indirect interactions through adjacent residues involved in heme binding. This work expands the toolbox of protein stapling strategies available for protein stabilization.

2.
J Biol Chem ; 296: 100107, 2021.
Article in English | MEDLINE | ID: mdl-33219127

ABSTRACT

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen-deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the 'DFD patch'), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.


Subject(s)
Adenosine Triphosphate/metabolism , Iron-Sulfur Proteins/metabolism , Adenosine Triphosphate/chemistry , Catalysis , Iron-Sulfur Proteins/chemistry , Mass Spectrometry , Nitrogenase/chemistry , Nitrogenase/metabolism , Photosynthesis , Protochlorophyllide/chemistry , Protochlorophyllide/metabolism , Substrate Specificity
3.
Proc Natl Acad Sci U S A ; 114(9): 2265-2270, 2017 02 28.
Article in English | MEDLINE | ID: mdl-28196882

ABSTRACT

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.


Subject(s)
Gene Products, tat/chemistry , High-Throughput Screening Assays , Phosphotransferases/chemistry , beta-Lactamases/chemistry , Amino Acid Substitution , Aspergillus niger/chemistry , Aspergillus niger/enzymology , Binding Sites , Escherichia coli/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Gene Products, tat/metabolism , HIV/chemistry , HIV/metabolism , Models, Molecular , Mutation , Peptide Library , Phosphotransferases/genetics , Phosphotransferases/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Stability , Protein Transport , Solubility , Structure-Activity Relationship , Two-Hybrid System Techniques , beta-Lactamases/genetics , beta-Lactamases/metabolism
4.
Proc Natl Acad Sci U S A ; 112(18): 5673-8, 2015 May 05.
Article in English | MEDLINE | ID: mdl-25902526

ABSTRACT

Human carbonic anhydrase II (HCA II) uses a Zn-bound OH(-)/H2O mechanism to catalyze the reversible hydration of CO2. This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pKa compared with free tyrosine. To further probe the pKa of this residue, NMR spectroscopic measurements of [(13)C]Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pKa of Tyr7 and how pH and Tyr proximity to Zn affect hydrogen-bonding interactions.


Subject(s)
Carbonic Anhydrases/chemistry , Crystallography, X-Ray/methods , Magnetic Resonance Spectroscopy/methods , Neutrons , Tyrosine/chemistry , Catalysis , Catalytic Domain , Cations , Enzymes/chemistry , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Protein Conformation , Protons , Static Electricity , Water/chemistry
5.
Biochemistry ; 56(20): 2529-2532, 2017 05 23.
Article in English | MEDLINE | ID: mdl-28481095

ABSTRACT

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. Both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). In the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND2 and ND-, suggesting a role for the copper ion in shifting the pKa of the amino terminus.


Subject(s)
Copper/chemistry , Mixed Function Oxygenases/chemistry , Oxygen/chemistry , Polysaccharides/chemistry , Catalytic Domain , Crystallography, X-Ray , Protein Conformation , Protons
6.
J Biol Chem ; 290(44): 26638-48, 2015 Oct 30.
Article in English | MEDLINE | ID: mdl-26354439

ABSTRACT

The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-ß-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.


Subject(s)
Cellulose/chemistry , Fungal Proteins/chemistry , Glucose-6-Phosphate/chemistry , Glucose/analogs & derivatives , Lipomyces/chemistry , Phosphotransferases/chemistry , Biocatalysis , Biomass , Cellulose/metabolism , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression , Glucose/chemistry , Glucose/metabolism , Glucose-6-Phosphate/metabolism , Kinetics , Lipomyces/enzymology , Magnesium/chemistry , Magnesium/metabolism , Manganese/chemistry , Manganese/metabolism , Models, Molecular , Phosphorylation , Phosphotransferases/genetics , Phosphotransferases/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
7.
IUBMB Life ; 68(9): 700-8, 2016 09.
Article in English | MEDLINE | ID: mdl-27416973

ABSTRACT

As methods for the use of anhydrosugars in chemical and biofuel production continue to develop, our collective knowledge of anhydrosugar processing enzymes continues to improve, including their mechanistic details, structural dynamics and modes of substrate binding. Of particular interest, anhydrosugar kinases, such as levoglucosan kinase (LGK) and 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), utilize an unusual mechanism whereby the sugar substrate is both cleaved and phosphorylated. The phosphorylated sugar can then be routed to other metabolic pathways, thereby allowing its further bioconversion. Advanced engineering efforts to improve the catalytic efficiency and stability of LGK have been steadily progressing. Other enzymes that cleave the glycosidic bond of disaccharide sugars containing an anhydrosugar component are also being identified and characterized. Accordingly, the potential future use of these enzymes in large-scale production strategies is becoming increasingly viable. Here, a mini-review of the observed characteristics of anhydrosugar processing enzymes is presented along with recent developments in the bioconversion of these sugars. © 2016 IUBMB Life 68(9):700-708, 2016.


Subject(s)
Escherichia coli/enzymology , Phosphotransferases/genetics , Sugar Alcohol Dehydrogenases/genetics , Biofuels , Carbohydrates/chemistry , Carbohydrates/genetics , Escherichia coli/genetics , Muramic Acids/chemistry , Muramic Acids/metabolism , Phosphorylation , Phosphotransferases/chemistry , Sugar Alcohol Dehydrogenases/chemistry , Sugar Alcohol Dehydrogenases/metabolism
8.
J Biol Chem ; 289(7): 4504-14, 2014 Feb 14.
Article in English | MEDLINE | ID: mdl-24362022

ABSTRACT

Anhydro-sugar kinases are unique from other sugar kinases in that they must cleave the 1,6-anhydro ring of their sugar substrate to phosphorylate it using ATP. Here we show that the peptidoglycan recycling enzyme 1,6-anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas aeruginosa undergoes large conformational changes during its catalytic cycle, with its two domains rotating apart by up to 32° around two hinge regions to expose an active site cleft into which the substrates 1,6-anhydroMurNAc and ATP can bind. X-ray structures of the open state bound to a nonhydrolyzable ATP analog (AMPPCP) and 1,6-anhydroMurNAc provide detailed insight into a ternary complex that forms preceding an operative Michaelis complex. Structural analysis of the hinge regions demonstrates a role for nucleotide binding and possible cross-talk between the bound ligands to modulate the opening and closing of AnmK. Although AnmK was found to exhibit similar binding affinities for ATP, ADP, and AMPPCP according to fluorescence spectroscopy, small angle x-ray scattering analyses revealed that AnmK adopts an open conformation in solution in the absence of ligand and that it remains in this open state after binding AMPPCP, as we had observed for our crystal structure of this complex. In contrast, the enzyme favored a closed conformation when bound to ADP in solution, consistent with a previous crystal structure of this complex. Together, our findings show that the open conformation of AnmK facilitates binding of both the sugar and nucleotide substrates and that large structural rearrangements must occur upon closure of the enzyme to correctly align the substrates and residues of the enzyme for catalysis.


Subject(s)
Adenosine Triphosphate/chemistry , Bacterial Proteins/chemistry , Muramic Acids/chemistry , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Pseudomonas aeruginosa/enzymology , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalysis , Crystallography, X-Ray , Muramic Acids/metabolism , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Binding , Protein Structure, Tertiary , Pseudomonas aeruginosa/genetics
9.
Proc Natl Acad Sci U S A ; 108(6): 2222-7, 2011 Feb 08.
Article in English | MEDLINE | ID: mdl-21245344

ABSTRACT

The attachment of ubiquitin (Ub) and the Ub-like (Ubl) molecule interferon-stimulated gene 15 (ISG15) to cellular proteins mediates important innate antiviral responses. Ovarian tumor (OTU) domain proteases from nairoviruses and arteriviruses were recently found to remove these molecules from host proteins, which inhibits Ub and ISG15-dependent antiviral pathways. This contrasts with the Ub-specific activity of known eukaryotic OTU-domain proteases. Here we describe crystal structures of a viral OTU domain from the highly pathogenic Crimean-Congo haemorrhagic fever virus (CCHFV) bound to Ub and to ISG15 at 2.5-Å and 2.3-Å resolution, respectively. The complexes provide a unique structural example of ISG15 bound to another protein and reveal the molecular mechanism of an ISG15 cross-reactive deubiquitinase. To accommodate structural differences between Ub and ISG15, the viral protease binds the ß-grasp folds of Ub and C-terminal Ub-like domain of ISG15 in an orientation that is rotated nearly 75° with respect to that observed for Ub bound to a representative eukaryotic OTU domain from yeast. Distinct structural determinants necessary for binding either substrate were identified and allowed the reengineering of the viral OTU protease into enzymes with increased substrate specificity, either for Ub or for ISG15. Our findings now provide the basis to determine in vivo the relative contributions of deubiquitination and deISGylation to viral immune evasion tactics, and a structural template of a promiscuous deubiquitinase from a haemorrhagic fever virus that can be targeted for inhibition using small-molecule-based strategies.


Subject(s)
Cytokines/chemistry , Hemorrhagic Fever Virus, Crimean-Congo/enzymology , Peptide Hydrolases/chemistry , Ubiquitins/chemistry , Viral Proteins/chemistry , Crystallography, X-Ray , Cytokines/genetics , Cytokines/immunology , Cytokines/metabolism , Female , Hemorrhagic Fever Virus, Crimean-Congo/genetics , Hemorrhagic Fever Virus, Crimean-Congo/immunology , Humans , Ovarian Neoplasms , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin/immunology , Ubiquitin/metabolism , Ubiquitins/genetics , Ubiquitins/immunology , Ubiquitins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
10.
Chembiochem ; 14(15): 1973-81, 2013 Oct 11.
Article in English | MEDLINE | ID: mdl-24009110

ABSTRACT

The increasing incidence of inducible chromosomal AmpC ß-lactamases within the clinic is a growing concern because these enzymes deactivate a broad range of even the most recently developed ß-lactam antibiotics. As a result, new strategies are needed to block the action of this antibiotic resistance enzyme. Presented here is a strategy to combat the action of inducible AmpC by inhibiting the ß-glucosaminidase NagZ, which is an enzyme involved in regulating the induction of AmpC expression. A divergent route facilitating the rapid synthesis of a series of N-acyl analogues of 2-acetamido-2-deoxynojirimycin is reported here. Among these compounds are potent NagZ inhibitors that are selective against functionally related human enzymes. These compounds reduce minimum inhibitory concentration values for ß-lactams against a clinically relevant Gram-negative bacterium bearing inducible chromosomal AmpC ß-lactamase, Pseudomonas aeruginosa. The structure of a NagZ-inhibitor complex provides insight into the molecular basis for inhibition by these compounds.


Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Discovery , Enzyme Inhibitors/pharmacology , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/enzymology , Hexosaminidases/antagonists & inhibitors , beta-Lactams/pharmacology , Hexosaminidases/chemistry , Hexosaminidases/metabolism , Humans , Microbial Sensitivity Tests , Models, Molecular , Peptidoglycan/metabolism , Protein Conformation
11.
Nat Commun ; 14(1): 7985, 2023 Dec 02.
Article in English | MEDLINE | ID: mdl-38042860

ABSTRACT

Hemoproteins have recently emerged as promising biocatalysts for new-to-nature carbene transfer reactions. However, mechanistic understanding of the interplay between productive and unproductive pathways in these processes is limited. Using spectroscopic, structural, and computational methods, we investigate the mechanism of a myoglobin-catalyzed cyclopropanation reaction with diazoketones. These studies shed light on the nature and kinetics of key catalytic steps in this reaction, including the formation of an early heme-bound diazo complex intermediate, the rate-determining nature of carbene formation, and the cyclopropanation mechanism. Our analyses further reveal the existence of a complex mechanistic manifold for this reaction that includes a competing pathway resulting in the formation of an N-bound carbene adduct of the heme cofactor, which was isolated and characterized by X-ray crystallography, UV-Vis, and Mössbauer spectroscopy. This species can regenerate the active biocatalyst, constituting a non-productive, yet non-destructive detour from the main catalytic cycle. These findings offer a valuable framework for both mechanistic analysis and design of hemoprotein-catalyzed carbene transfer reactions.


Subject(s)
Methane , Myoglobin , Myoglobin/chemistry , Catalysis , Methane/chemistry , Heme
12.
J Biol Chem ; 286(14): 12283-91, 2011 Apr 08.
Article in English | MEDLINE | ID: mdl-21288904

ABSTRACT

Anhydro-N-acetylmuramic acid kinase (AnmK) catalyzes the ATP-dependent conversion of the Gram-negative peptidoglycan (PG) recycling intermediate 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) to N-acetylmuramic acid-6-phosphate (MurNAc-6-P). Here we present crystal structures of Pseudomonas aeruginosa AnmK in complex with its natural substrate, anhMurNAc, and a product of the reaction, ADP. AnmK is homodimeric, with each subunit comprised of two subdomains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. The conversion of anhMurNAc to MurNAc-6-P involves both cleavage of the 1,6-anhydro ring of anhMurNAc along with addition of a phosphoryl group to O6 of the sugar, and thus represents an unusual enzymatic mechanism involving the formal addition of H3PO4 to anhMurNAc. The structural complexes and NMR analysis of the reaction suggest that a water molecule, activated by Asp-182, attacks the anomeric carbon of anhMurNAc, aiding cleavage of the 1,6-anhydro bond and facilitating the capture of the γ phosphate of ATP by O6 via an in-line phosphoryl transfer. AnmK is active only against anhMurNAc and not the metabolically related 1,6-anhydro-N-acetylmuramyl peptides, suggesting that the cytosolic N-acetyl-anhydromuramyl-l-alanine amidase AmpD must first remove the stem peptide from these PG muropeptide catabolites before anhMurNAc can be acted upon by AnmK. Our studies provide the foundation for a mechanistic model for the dual activities of AnmK as a hydrolase and a kinase of an unusual heterocyclic monosaccharide.


Subject(s)
Bacterial Proteins/metabolism , Muramic Acids/metabolism , Phosphotransferases/metabolism , Pseudomonas aeruginosa/enzymology , Adenosine Triphosphate , Bacterial Proteins/genetics , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Mutagenesis, Site-Directed , Phosphotransferases/genetics , Protein Structure, Secondary , Pseudomonas aeruginosa/genetics
13.
PLoS One ; 17(11): e0277670, 2022.
Article in English | MEDLINE | ID: mdl-36395154

ABSTRACT

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.


Subject(s)
Alanine Dehydrogenase , Mycobacterium tuberculosis , Alanine Dehydrogenase/metabolism , Mycobacterium tuberculosis/metabolism , Nucleosides , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Drug Discovery
14.
J Biol Chem ; 285(26): 20273-80, 2010 Jun 25.
Article in English | MEDLINE | ID: mdl-20368332

ABSTRACT

E1 ubiquitin-activating enzymes (UBAs) are large multidomain proteins that catalyze formation of a thioester bond between the terminal carboxylate of a ubiquitin or ubiquitin-like modifier (UBL) and a conserved cysteine in an E2 protein, producing reactive ubiquityl units for subsequent ligation to substrate lysines. Two important E1 reaction intermediates have been identified: a ubiquityl-adenylate phosphoester and a ubiquityl-enzyme thioester. However, the mechanism of thioester bond formation and its subsequent transfer to an E2 enzyme remains poorly understood. We have determined the crystal structure of the human UFM1 (ubiquitin-fold modifier 1) E1-activating enzyme UBA5, bound to ATP, revealing a structure that shares similarities with both large canonical E1 enzymes and smaller ancestral E1-like enzymes. In contrast to other E1 active site cysteines, which are in a variably sized domain that is separate and flexible relative to the adenylation domain, the catalytic cysteine of UBA5 (Cys(250)) is part of the adenylation domain in an alpha-helical motif. The novel position of the UBA5 catalytic cysteine and conformational changes associated with ATP binding provides insight into the possible mechanisms through which the ubiquityl-enzyme thioester is formed. These studies reveal structural features that further our understanding of the UBA5 enzyme reaction mechanism and provide insight into the evolution of ubiquitin activation.


Subject(s)
Adenosine Triphosphate/metabolism , Ubiquitin-Activating Enzymes/chemistry , Ubiquitin-Activating Enzymes/metabolism , Amino Acid Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Proteins , Sequence Homology, Amino Acid , Ubiquitin-Activating Enzymes/genetics
15.
ACS Catal ; 9(2): 1514-1524, 2019 Feb 01.
Article in English | MEDLINE | ID: mdl-31134138

ABSTRACT

Recent advances in metalloprotein engineering have led to the development of a myoglobin-based catalyst, Mb(H64V,V68A), capable of promoting the cyclopropanation of vinylarenes with high efficiency and high diastereo- and enantioselectivity. Whereas many enzymes evolved in nature often exhibit catalytic proficiency and exquisite stereoselectivity, how these features are achieved for a non-natural reaction has remained unclear. In this work, the structural determinants responsible for chiral induction and high stereocontrol in Mb(H64V,V68A)-catalyzed cyclopropanation were investigated via a combination of crystallographic, computational (DFT), and structure-activity analyses. Our results show the importance of steric complementarity and non-covalent interactions involving first-sphere active site residues, heme-carbene, and the olefin substrate, in dictating the stereochemical outcome of the cyclopropanation reaction. High stereocontrol is achieved through two major mechanisms. First, by enforcing a specific conformation of the heme-bound carbene within the active site. Second, by controlling the geometry of attack of the olefin on the carbene via steric occlusion, attractive van der Waals forces and protein-mediated π-π interactions with the olefin substrate. These insights could be leveraged to expand the substrate scope of the myoglobin-based cyclopropanation catalyst toward non-activated olefins and to increase its cyclopropanation activity in the presence of a bulky α-diazo-ester. This work sheds first light into the origin of enzyme-catalyzed enantioselective cyclopropanation, furnishing a mechanistic framework for both understanding the reactivity of current systems and guiding the future development of biological catalysts for this class of synthetically important, abiotic transformations.

16.
Nat Commun ; 10(1): 2653, 2019 06 14.
Article in English | MEDLINE | ID: mdl-31201319

ABSTRACT

Ribonucleotide reductases (RNRs) use a conserved radical-based mechanism to catalyze the conversion of ribonucleotides to deoxyribonucleotides. Within the RNR family, class Ib RNRs are notable for being largely restricted to bacteria, including many pathogens, and for lacking an evolutionarily mobile ATP-cone domain that allosterically controls overall activity. In this study, we report the emergence of a distinct and unexpected mechanism of activity regulation in the sole RNR of the model organism Bacillus subtilis. Using a hypothesis-driven structural approach that combines the strengths of small-angle X-ray scattering (SAXS), crystallography, and cryo-electron microscopy (cryo-EM), we describe the reversible interconversion of six unique structures, including a flexible active tetramer and two inhibited helical filaments. These structures reveal the conformational gymnastics necessary for RNR activity and the molecular basis for its control via an evolutionarily convergent form of allostery.


Subject(s)
Allosteric Site/genetics , Bacterial Proteins/genetics , Ribonucleotide Reductases/genetics , Allosteric Regulation/genetics , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , Evolution, Molecular , Models, Molecular , Protein Structure, Quaternary/genetics , Ribonucleotide Reductases/chemistry , Ribonucleotide Reductases/metabolism , Ribonucleotide Reductases/ultrastructure , Ribonucleotides/metabolism , Scattering, Small Angle
17.
J Mol Biol ; 365(5): 1545-58, 2007 Feb 02.
Article in English | MEDLINE | ID: mdl-17137595

ABSTRACT

Glutaredoxins act as reducing agents for the large subunit of ribonucleotide reductase (R1) in many prokaryotes and eukaryotes, including humans. The same relationship has been proposed for the glutaredoxin and R1 proteins expressed by all orthopoxviruses, including vaccinia, variola, and ectromelia virus. Interestingly, the orthopoxviral proteins share 45% and 78% sequence identity with human glutaredoxin-1 (Grx-1) and R1, respectively. To study structure-function relationships of the vertebrate Grx-1 family, and reveal potential viral adaptations, we have determined crystal structures of the ectromelia virus glutaredoxin, EVM053, in the oxidized and reduced states. The structures show a large redox-induced conformational rearrangement of Tyr21 and Thr22 near the active site. We predict that the movement of Tyr21 is a viral-specific adaptation that increases the redox potential by stabilizing the reduced state. The conformational switch of Thr22 appears to be shared by vertebrate Grx-1 and may affect the strictly conserved Lys20. A crystal packing-induced structural change in residues 68-70 affects the GSH-binding loop, and our structures reveal a potential interaction network that connects the GSH-binding loop and the active site. EVM053 also exhibits a novel cis-proline (Pro53) in a loop that has been shown to contribute to R1-binding in Escherichia coli Grx-1. The cis-peptide bond of Pro53 may be required to promote electrostatic interactions between Lys52 and the C-terminal carboxylate of R1. Finally, dimethylarsenite was covalently attached to Cys23 in one reduced EVM053 structure and our preliminary data show that EVM053 has dimethylarsenate reductase activity.


Subject(s)
Ectromelia virus/chemistry , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Animals , Binding Sites , Crystallography, X-Ray , Glutaredoxins , Glutathione/metabolism , Humans , Models, Molecular , Oxidation-Reduction , Protein Binding , Protein Structure, Secondary , Protein Subunits/metabolism , Ribonucleotide Reductases/metabolism , Static Electricity , Structure-Activity Relationship , Swine , Threonine/chemistry , X-Ray Diffraction
18.
FEBS Lett ; 590(1): 34-42, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26763108

ABSTRACT

Lytic polysaccharide monooxygenases (LPMOs) boost enzymatic depolymerization of recalcitrant polysaccharides, such as chitin and cellulose. We have studied a chitin-active LPMO domain (JdLPMO10A) that is considerably smaller (15.5 kDa) than all structurally characterized LPMOs so far and that is part of a modular protein containing a GH18 chitinase. The 1.55 Å resolution structure revealed deletions of interacting loops that protrude from the core ß-sandwich scaffold in larger LPMO10s. Despite these deletions, the enzyme is active on alpha- and beta-chitin, and the chitin-binding surface previously described for larger LPMOs is fully conserved. JdLPMO10A may represent a minimal scaffold needed to catalyse the powerful LPMO reaction.


Subject(s)
Actinobacteria/enzymology , Bacterial Proteins/metabolism , Cellulose/metabolism , Chitin/metabolism , Chitinases/metabolism , Mixed Function Oxygenases/metabolism , Models, Molecular , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Catalytic Domain , Cellulose/chemistry , Chitin/chemistry , Chitinases/chemistry , Conserved Sequence , Crystallography, X-Ray , Hydrolysis , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/genetics , Molecular Weight , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Phylogeny , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structural Homology, Protein , Substrate Specificity
19.
Article in English | MEDLINE | ID: mdl-16511093

ABSTRACT

Ectromelia, vaccinia, smallpox and other closely related viruses of the orthopoxvirus genus encode a glutaredoxin gene that is not present in poxviruses outside of this genus. The vaccinia glutaredoxin O2L has been implicated as the reducing agent for ribonucleotide reductase and may thus play an important role in viral deoxyribonucleotide synthesis. As part of an effort to understand nucleotide metabolism by poxviruses, EVM053, the O2L ortholog of the ectromelia virus, has been crystallized. EVM053 crystallizes in space group C222(1), with unit-cell parameters a = 61.98, b = 67.57, c = 108.55 A. Diffraction data have been processed to 1.8 A resolution and a self-rotation function indicates that there are two molecules per asymmetric unit.


Subject(s)
Ectromelia virus/chemistry , Oxidoreductases/chemistry , Cloning, Molecular , Crystallization/methods , Glutaredoxins , Nucleotides/metabolism , Oxidoreductases/genetics , Oxidoreductases/isolation & purification , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/isolation & purification , X-Ray Diffraction
20.
ACS Synth Biol ; 4(11): 1235-43, 2015 Nov 20.
Article in English | MEDLINE | ID: mdl-26369947

ABSTRACT

Synthetic metabolic pathways often suffer from low specific productivity, and new methods that quickly assess pathway functionality for many thousands of variants are urgently needed. Here we present an approach that enables the rapid and parallel determination of sequence effects on flux for complete gene-encoding sequences. We show that this method can be used to determine the effects of over 8000 single point mutants of a pyrolysis oil catabolic pathway implanted in Escherichia coli. Experimental sequence-function data sets predicted whether fitness-enhancing mutations to the enzyme levoglucosan kinase resulted from enhanced catalytic efficiency or enzyme stability. A structure of one design incorporating 38 mutations elucidated the structural basis of high fitness mutations. One design incorporating 15 beneficial mutations supported a 15-fold improvement in growth rate and greater than 24-fold improvement in enzyme activity relative to the starting pathway. This technique can be extended to improve a wide variety of designed pathways.


Subject(s)
Escherichia coli/metabolism , Glucose/analogs & derivatives , Biocatalysis , Biomass , Enzyme Stability , Escherichia coli/enzymology , Glucose/metabolism , Phosphotransferases/genetics , Phosphotransferases/metabolism , Synthetic Biology
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