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4.
Faraday Discuss ; 2024 Jul 24.
Article in English | MEDLINE | ID: mdl-39046423

ABSTRACT

Enzymes are being engineered to catalyze chemical reactions for many practical applications in chemistry and biotechnology. The approaches used are surveyed in this short review, emphasizing methods for accessing reactivities not expressed by native protein scaffolds. The successful generation of completely de novo enzymes that rival the rates and selectivities of their natural counterparts highlights the potential role that designer enzymes may play in the coming years in research, industry, and medicine. Some challenges that need to be addressed to realize this ambitious dream are considered together with possible solutions.

5.
Nat Commun ; 15(1): 3576, 2024 Apr 27.
Article in English | MEDLINE | ID: mdl-38678040

ABSTRACT

Controlled assembly of a protein shell around a viral genome is a key step in the life cycle of many viruses. Here we report a strategy for regulating the co-assembly of nonviral proteins and nucleic acids into highly ordered nucleocapsids in vitro. By fusing maltose binding protein to the subunits of NC-4, an engineered protein cage that encapsulates its own encoding mRNA, we successfully blocked spontaneous capsid assembly, allowing isolation of the individual monomers in soluble form. To initiate RNA-templated nucleocapsid formation, the steric block can be simply removed by selective proteolysis. Analyses by transmission and cryo-electron microscopy confirmed that the resulting assemblies are structurally identical to their RNA-containing counterparts produced in vivo. Enzymatically triggered cage formation broadens the range of RNA molecules that can be encapsulated by NC-4, provides unique opportunities to study the co-assembly of capsid and cargo, and could be useful for studying other nonviral and viral assemblies.


Subject(s)
Cryoelectron Microscopy , Maltose-Binding Proteins , Nucleocapsid , Nucleocapsid/metabolism , Nucleocapsid/ultrastructure , Maltose-Binding Proteins/metabolism , Maltose-Binding Proteins/genetics , Virus Assembly , Capsid/metabolism , RNA, Viral/metabolism , RNA, Viral/genetics , Capsid Proteins/metabolism , Capsid Proteins/genetics , Capsid Proteins/chemistry , RNA, Messenger/metabolism , RNA, Messenger/genetics
6.
Nat Chem Biol ; 20(6): 761-769, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38308044

ABSTRACT

Engineered biosynthetic assembly lines could revolutionize the sustainable production of bioactive natural product analogs. Although yeast display is a proven, powerful tool for altering the substrate specificity of gatekeeper adenylation domains in nonribosomal peptide synthetases (NRPSs), comparable strategies for other components of these megaenzymes have not been described. Here we report a high-throughput approach for engineering condensation (C) domains responsible for peptide elongation. We show that a 120-kDa NRPS module, displayed in functional form on yeast, can productively interact with an upstream module, provided in solution, to produce amide products tethered to the yeast surface. Using this system to screen a large C-domain library, we reprogrammed a surfactin synthetase module to accept a fatty acid donor, increasing catalytic efficiency for this noncanonical substrate >40-fold. Because C domains can function as selectivity filters in NRPSs, this methodology should facilitate the precision engineering of these molecular assembly lines.


Subject(s)
Peptide Synthases , Peptide Synthases/metabolism , Peptide Synthases/genetics , Peptide Synthases/chemistry , Substrate Specificity , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Protein Engineering/methods , High-Throughput Screening Assays , Protein Domains
7.
Acta Biomater ; 177: 107-117, 2024 03 15.
Article in English | MEDLINE | ID: mdl-38382830

ABSTRACT

Designing proteins that fold and assemble over different length scales provides a way to tailor the mechanical properties and biological performance of hydrogels. In this study, we designed modular proteins that self-assemble into fibrillar networks and, as a result, form hydrogel materials with novel properties. We incorporated distinct functionalities by connecting separate self-assembling (A block) and cell-binding (B block) domains into single macromolecules. The number of self-assembling domains affects the rigidity of the fibers and the final storage modulus G' of the materials. The mechanical properties of the hydrogels could be tuned over a broad range (G' = 0.1 - 10 kPa), making them suitable for the cultivation and differentiation of multiple cell types, including cortical neurons and human mesenchymal stem cells. Moreover, we confirmed the bioavailability of cell attachment domains in the hydrogels that can be further tailored for specific cell types or other biological applications. Finally, we demonstrate the versatility of the designed proteins for application in biofabrication as 3D scaffolds that support cell growth and guide their function. STATEMENT OF SIGNIFICANCE: Designed proteins that enable the decoupling of biophysical and biochemical properties within the final material could enable modular biomaterial engineering. In this context, we present a designed modular protein platform that integrates self-assembling domains (A blocks) and cell-binding domains (B blocks) within a single biopolymer. The linking of assembly domains and cell-binding domains this way provided independent tuning of mechanical properties and inclusion of biofunctional domains. We demonstrate the use of this platform for biofabrication, including neural cell culture and 3D printing of scaffolds for mesenchymal stem cell culture and differentiation. Overall, this work highlights how informed design of biopolymer sequences can enable the modular design of protein-based hydrogels with independently tunable biophysical and biochemical properties.


Subject(s)
Hydrogels , Mesenchymal Stem Cells , Humans , Hydrogels/chemistry , Proteins/chemistry , Biocompatible Materials/metabolism , Biopolymers , Tissue Engineering
8.
J Am Chem Soc ; 146(3): 1957-1966, 2024 Jan 24.
Article in English | MEDLINE | ID: mdl-38264790

ABSTRACT

Nitrene transfer reactions catalyzed by heme proteins have broad potential for the stereoselective formation of carbon-nitrogen bonds. However, competition between productive nitrene transfer and the undesirable reduction of nitrene precursors limits the broad implementation of such biocatalytic methods. Here, we investigated the reduction of azides by the model heme protein myoglobin to gain mechanistic insights into the factors that control the fate of key reaction intermediates. In this system, the reaction proceeds via a proposed nitrene intermediate that is rapidly reduced and protonated to give a reactive ferrous amide species, which we characterized by UV/vis and Mössbauer spectroscopies, quantum mechanical calculations, and X-ray crystallography. Rate-limiting protonation of the ferrous amide to produce the corresponding amine is the final step in the catalytic cycle. These findings contribute to our understanding of the heme protein-catalyzed reduction of azides and provide a guide for future enzyme engineering campaigns to create more efficient nitrene transferases. Moreover, harnessing the reduction reaction in a chemoenzymatic cascade provided a potentially practical route to substituted pyrroles.

9.
Angew Chem Int Ed Engl ; 62(49): e202315565, 2023 Dec 04.
Article in English | MEDLINE | ID: mdl-37933996

ABSTRACT

Albert Eschenmoser, one of the greatest organic chemists of the past hundred years, died on July 14, 2023 at the age of 97. The extraordinary breadth of his scientific contributions ranged from synthetic methodology, structure elucidation, and synthesis of natural products to the chemical etiology of biomolecular structures.

10.
ACS Chem Biol ; 18(12): 2516-2523, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-37983914

ABSTRACT

Nonribosomal peptides constitute an important class of natural products that display a wide range of bioactivities. They are biosynthesized by large assembly lines called nonribosomal peptide synthetases (NRPSs). Engineering NRPS modules represents an attractive strategy for generating customized synthetases for the production of peptide variants with improved properties. Here, we explored the yeast display of NRPS elongation and termination modules as a high-throughput screening platform for assaying adenylation domain activity and altering substrate specificity. Depending on the module, display of A-T bidomains or C-A-T tridomains, which also include an upstream condensation domain, proved to be most effective. Reprograming a tyrocidine synthetase elongation module to accept 4-propargyloxy-phenylalanine, a noncanonical amino acid that is not activated by the native protein, illustrates the utility of this approach for altering NRPS specificity at internal sites.


Subject(s)
Peptide Synthases , Yeasts , Peptide Synthases/metabolism , Phenylalanine , Peptides/chemistry
11.
J Mater Chem B ; 11(28): 6540-6546, 2023 07 19.
Article in English | MEDLINE | ID: mdl-37427706

ABSTRACT

Charge-driven inclusion complex formation in live cells was examined using a degradation-prone fluorescent protein and a series of protein cages. The results show that sufficiently strong host-guest ionic interaction and an intact shell-like structure are crucial for the protective guest encapsulation.

12.
Nat Prod Rep ; 40(9): 1479-1497, 2023 09 20.
Article in English | MEDLINE | ID: mdl-37231979

ABSTRACT

Covering: 1878 to early 2023Cyanophycin is a biopolymer consisting of a poly-aspartate backbone with arginines linked to each Asp sidechain through isopeptide bonds. Cyanophycin is made by cyanophycin synthetase 1 or 2 through ATP-dependent polymerization of Asp and Arg, or ß-Asp-Arg, respectively. It is degraded into dipeptides by exo-cyanophycinases, and these dipeptides are hydrolyzed into free amino acids by general or dedicated isodipeptidase enzymes. When synthesized, chains of cyanophycin coalesce into large, inert, membrane-less granules. Although discovered in cyanobacteria, cyanophycin is made by species throughout the bacterial kingdom, and cyanophycin metabolism provides advantages for toxic bloom forming algae and some human pathogens. Some bacteria have developed dedicated schemes for cyanophycin accumulation and use, which include fine temporal and spatial regulation. Cyanophycin has also been heterologously produced in a variety of host organisms to a remarkable level, over 50% of the host's dry mass, and has potential for a variety of green industrial applications. In this review, we summarize the progression of cyanophycin research, with an emphasis on recent structural studies of enzymes in the cyanophycin biosynthetic pathway. These include several unexpected revelations that show cyanophycin synthetase to be a very cool, multi-functional macromolecular machine.


Subject(s)
Bacterial Proteins , Cyanobacteria , Humans , Bacterial Proteins/metabolism , Cyanobacteria/metabolism , Peptide Synthases/metabolism , Dipeptides/chemistry
13.
Protein Sci ; 32(7): e4685, 2023 07.
Article in English | MEDLINE | ID: mdl-37222490

ABSTRACT

Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide ß-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized.


Subject(s)
Cyanobacteria , Peptide Synthases , Peptide Synthases/chemistry , Aspartic Acid , Bacterial Proteins/chemistry
14.
ACS Appl Bio Mater ; 6(2): 591-602, 2023 02 20.
Article in English | MEDLINE | ID: mdl-36626688

ABSTRACT

Diagnostic medical imaging utilizes magnetic resonance (MR) to provide anatomical, functional, and molecular information in a single scan. Nanoparticles are often labeled with Gd(III) complexes to amplify the MR signal of contrast agents (CAs) with large payloads and high proton relaxation efficiencies (relaxivity, r1). This study examined the MR performance of two structurally unique cages, AaLS-13 and OP, labeled with Gd(III). The cages have characteristics relevant for the development of theranostic platforms, including (i) well-defined structure, symmetry, and size; (ii) the amenability to extensive engineering; (iii) the adjustable loading of therapeutically relevant cargo molecules; (iv) high physical stability; and (v) facile manufacturing by microbial fermentation. The resulting conjugates showed significantly enhanced proton relaxivity (r1 = 11-18 mM-1 s-1 at 1.4 T) compared to the Gd(III) complex alone (r1 = 4 mM-1 s-1). Serum phantom images revealed 107% and 57% contrast enhancements for Gd(III)-labeled AaLS-13 and OP cages, respectively. Moreover, proton nuclear magnetic relaxation dispersion (1H NMRD) profiles showed maximum relaxivity values of 50 mM-1 s-1. Best-fit analyses of the 1H NMRD profiles attributed the high relaxivity of the Gd(III)-labeled cages to the slow molecular tumbling of the conjugates and restricted local motion of the conjugated Gd(III) complex.


Subject(s)
Nanoparticles , Protons , Contrast Media/chemistry , Gadolinium/chemistry , Magnetic Resonance Imaging/methods
15.
Protein Sci ; 31(11): e4405, 2022 11.
Article in English | MEDLINE | ID: mdl-36305767

ABSTRACT

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded ß-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds.


Subject(s)
Protein Engineering , Proteins , Proteins/genetics , Protein Engineering/methods
16.
J Am Chem Soc ; 144(38): 17567-17575, 2022 09 28.
Article in English | MEDLINE | ID: mdl-36070491

ABSTRACT

High-throughput engineering has the potential to revolutionize the customization of biosynthetic assembly lines for the sustainable production of pharmaceutically relevant natural product analogs. Here, we show that the substrate specificity of gatekeeper adenylation domains of nonribosomal peptide synthetases can be switched from an α-amino acid to an α-hydroxy acid in a single round of combinatorial mutagenesis and selection using yeast cell surface display. In addition to shedding light on how such proteins discriminate between amino and hydroxy groups, the remodeled domains function in a pathway context to produce α-hydroxy acid-containing linear peptides and cyclic depsipeptides with high efficiency. Site-specific replacement of backbone amines with oxygens by an engineered synthetase provides the means to probe and tune the activities of diverse peptide metabolites in a simple and predictable fashion.


Subject(s)
Biological Products , Depsipeptides , Amines , Amino Acids/metabolism , Hydroxy Acids , Peptide Synthases/metabolism , Substrate Specificity
17.
Chembiochem ; 23(20): e202200332, 2022 10 19.
Article in English | MEDLINE | ID: mdl-35951442

ABSTRACT

Although viruses have been successfully repurposed as vaccines, antibiotics, and anticancer therapeutics, they also raise concerns regarding genome integration and immunogenicity. Virus-like particles and non-viral protein cages represent a potentially safer alternative but often lack desired functionality. Here, we investigated the utility of a new enzymatic bioconjugation method, called lysine acylation using conjugating enzymes (LACE), to chemoenzymatically modify protein cages. We equipped two structurally distinct protein capsules with a LACE-reactive peptide tag and demonstrated their modification with diverse ligands. This modular approach combines the advantages of chemical conjugation and genetic fusion and allows for site-specific modification with recombinant proteins as well as synthetic peptides with facile control of the extent of labeling. This strategy has the potential to fine-tune protein containers of different shape and size by providing them with new properties that go beyond their biologically native functions.


Subject(s)
Lysine , Peptides , Lysine/metabolism , Peptides/metabolism , Recombinant Proteins/genetics , Acylation , Anti-Bacterial Agents
18.
Biochim Biophys Acta Gen Subj ; 1866(11): 130217, 2022 11.
Article in English | MEDLINE | ID: mdl-35905922

ABSTRACT

BACKGROUND: Cyanophycinases are serine protease family enzymes which are required for the metabolism of cyanophycin, the natural polymer multi-L-arginyl-poly(L-aspartic acid). Cyanophycinases degrade cyanophycin to ß-Asp-Arg dipeptides, which enables use of this important store of fixed nitrogen. METHODS: We used genetic code expansion to incorporate diaminopropionic acid into cyanophycinase in place of the active site serine, and determined a high-resolution structure of the covalent acyl-enzyme intermediate resulting from attack of cyanophycinase on a short cyanophycin segment. RESULTS: The structure indicates that cyanophycin dipeptide residues P1 and P1' bind shallow pockets adjacent to the catalytic residues. We observe many cyanophycinase - P1 dipeptide interactions in the co-complex structure. Calorimetry measurements show that at least two cyanophycin dipeptides are needed for high affinity binding to cyanophycinase. We also characterized a putative cyanophycinase which we found to be structurally very similar but that shows no activity and could not be activated by mutation of its active site. GENERAL SIGNIFICANCE: Despite its peptidic structure, cyanophycin is resistant to degradation by peptidases and other proteases. Our results help show how cyanophycinase can specifically bind and degrade this important polymer.


Subject(s)
Dipeptides , Peptide Hydrolases , Bacterial Proteins , Polymers
19.
Nat Commun ; 13(1): 3923, 2022 07 07.
Article in English | MEDLINE | ID: mdl-35798723

ABSTRACT

Cyanophycin is a nitrogen reserve biopolymer in many bacteria that has promising industrial applications. Made by cyanophycin synthetase 1 (CphA1), it has a poly-L-Asp backbone with L-Arg residues attached to each aspartate sidechain. CphA1s are thought to typically require existing segments of cyanophycin to act as primers for cyanophycin polymerization. In this study, we show that most CphA1s will not require exogenous primers and discover the surprising cause of primer independence: CphA1 can make minute quantities of cyanophycin without primer, and an unexpected, cryptic metallopeptidase-like active site in the N-terminal domain of many CphA1s digests these into primers, solving the problem of primer availability. We present co-complex cryo-EM structures, make mutations that transition CphA1s between primer dependence and independence, and demonstrate that primer dependence can be a limiting factor for cyanophycin production in heterologous hosts. In CphA1, domains with opposite catalytic activities combine into a remarkable, self-sufficient, biosynthetic nanomachine.


Subject(s)
Bacterial Proteins , Peptide Synthases , Bacterial Proteins/chemistry , Catalytic Domain , Peptide Synthases/metabolism , Plant Proteins/metabolism , Polymerization
20.
Nature ; 606(7912): 49-58, 2022 06.
Article in English | MEDLINE | ID: mdl-35650353

ABSTRACT

The ability to design efficient enzymes from scratch would have a profound effect on chemistry, biotechnology and medicine. Rapid progress in protein engineering over the past decade makes us optimistic that this ambition is within reach. The development of artificial enzymes containing metal cofactors and noncanonical organocatalytic groups shows how protein structure can be optimized to harness the reactivity of nonproteinogenic elements. In parallel, computational methods have been used to design protein catalysts for diverse reactions on the basis of fundamental principles of transition state stabilization. Although the activities of designed catalysts have been quite low, extensive laboratory evolution has been used to generate efficient enzymes. Structural analysis of these systems has revealed the high degree of precision that will be needed to design catalysts with greater activity. To this end, emerging protein design methods, including deep learning, hold particular promise for improving model accuracy. Here we take stock of key developments in the field and highlight new opportunities for innovation that should allow us to transition beyond the current state of the art and enable the robust design of biocatalysts to address societal needs.


Subject(s)
Biocatalysis , Biotechnology , Protein Engineering , Proteins , Biotechnology/methods , Biotechnology/trends , Protein Engineering/methods , Protein Engineering/trends , Proteins/chemistry , Proteins/metabolism
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