The enzyme mechanism of a de novo designed and evolved aldolase

The combination of computational enzyme design and laboratory evolution is a successful strategy for the development of biocatalysts with non-natural function, one example being the artificial retroaldolase RA95.1,2 This enzyme utilizes amine catalysis via a reactive lysine residue to cleave the unnatural aldol substrate methodol (Figure 1A). The low initial catalytic activity of the computational design was improved tremendously over many rounds of directed evolution, yielding an efficient biocatalyst for both aldol cleavage as well as synthesis with rate acceleration and stereoselectivity comparable to natural aldolases (Figure 1B).3,4 Key to this success was an ultrahigh-throughput (uHTP) screening technique applied for the late stages of optimization.4 In this work, we analyzed changes in enzyme mechanism along the evolutionary trajectory of RA95 that led to more efficient catalysis. To that end, we determined the rate-limiting step for different enzyme variants by probing individual steps of the aldolase mechanism kinetically. We found a shift towards product release being overall rate-limiting for aldol cleavage catalyzed by highly evolved variants of RA95. Specifically, the conversion between Schiff base and enamine intermediate formed from acetone, a (de-)protonation-dependent process, is the slowest step we probed. Our results indicate that uHTP screening is essential to efficiently evolve a multi-step enzyme mechanism, as it allows the optimization of several mechanistic steps in parallel. By comparing our findings to kinetic and structural studies on natural aldolases, we provide valuable feedback to improve future laboratory evolution approaches as well as the success rate of computational enzyme design. Please click Additional Files below to see the full abstract

Optimization of Enzyme Mechanism along the Evolutionary Trajectory of a Computationally Designed (Retro-)Aldolase

ISSN:0002-7863ISSN:1520-512

The Molecular Mechanism of Hsp100 Chaperone Inhibition by the Prion Curing Agent Guanidinium Chloride

The Hsp100 chaperones ClpB and Hsp104 utilize the energy from ATP hydrolysis to reactivate aggregated proteins in concert with the DnaK/Hsp70 chaperone system, thereby playing an important role in protein quality control. They belong to the family of AAA+ proteins (ATPases associated with various cellular activities), possess two nucleotide binding domains per monomer (NBD1 and NBD2), and oligomerize into hexameric ring complexes. Furthermore, Hsp104 is involved in yeast prion propagation and inheritance. It is well established that low concentrations of guanidinium chloride (GdmCl) inhibit the ATPase activity of Hsp104, leading to so called “prion curing,” the loss of prion-related phenotypes. Here, we present mechanistic details about the Hsp100 chaperone inhibition by GdmCl using the Hsp104 homolog ClpB from Thermus thermophilus. Initially, we demonstrate that NBD1 of ClpB, which was previously considered inactive as a separately expressed construct, is a fully active ATPase on its own. Next, we show that only NBD1, but not NBD2, is affected by GdmCl. We present a crystal structure of ClpB NBD1 in complex with GdmCl and ADP, showing that the Gdm(+) ion binds specifically to the active site of NBD1. A conserved essential glutamate residue is involved in this interaction. Additionally, Gdm(+) interacts directly with the nucleotide, thereby increasing the nucleotide binding affinity of NBD1. We propose that both the interference with the essential glutamate and the modulation of nucleotide binding properties in NBD1 is responsible for the GdmCl-specific inhibition of Hsp100 chaperones

Americium preferred: Lanmodulin, a natural lanthanide-binding protein favors an actinide over lanthanides

The separation and recycling of lanthanides is an active area of research with a growing demand that calls for more environmentally friendly lanthanide sources. Likewise, the efficient and industrial separation of lanthanides from the minor actinides (Np, Am - Fm) is one of the key questions for closing the nuclear fuel cycle; reducing costs and increasing safety. With the advent of the field of lanthanide dependent bacterial metabolism, bio-inspired applications are in reach. Here, we utilize the natural lanthanide chelator Lanmodulin and the luminescent probes Eu3+ and Cm3+ to investigate the inter-metal competition behavior of all lanthanides (except Pm) and four actinides (Np, Pu, Am, Cm) to Lanmodulin. Using time resolved laser induced fluorescence spectroscopy we show that Lanmodulin has the highest relative binding affinity to Nd3+ and Eu3+ among the lanthanide series. When equimolar mixtures of Cm3+ and Am3+ are added to Lanmodulin, Lanmodulin preferentially binds to Am3+ over Cm3+ whilst Nd3+ and Cm3+ bind with similar relative affinity. The results presented show that a natural lanthanide binding protein can bind various actinides with high relative affinity, paving the way to bio inspired separation applications. In addition, an easy and versatile method was developed, using the fluorescence properties of only two elements, Eu and Cm, for inter-metal competition studies regarding lanthanides and selected actinides and their binding to biological molecules

Coupling of Oligomerization and Nucleotide Binding in the AAA+ Chaperone ClpB

Members of the family of ATPases associated with various cellular activities (AAA+) typically form homohexameric ring complexes and are able to remodel their substrates, such as misfolded proteins or protein−protein complexes, in an ATP−driven process. The molecular mechanism by which ATP hydrolysis is coordinated within the multimeric complex and the energy is converted into molecular motions, however, is poorly understood. This is partly due to the fact that the oligomers formed by AAA+ proteins represent a highly complex system and analysis depends on simplification and prior knowledge. Here, we present nucleotide binding and oligomer assembly kinetics of the AAA+ protein ClpB, a molecular chaperone that is able to disaggregate protein aggregates in concert with the DnaK chaperone system. ClpB bears two AAA+ domains (NBD1 and NBD2) on one subunit and forms homohexameric ring complexes. In order to dissect individual mechanistic steps, we made use of a reconstituted system based on two individual constructs bearing either the N−terminal (NBD1) or the C−terminal AAA+ domain (NBD2). In contrast to the C−terminal construct, the N−terminal construct does not bind the fluorescent nucleotide MANT−dADP in isolation. However, sequential mixing experiments suggest that NBD1 obtains nucleotide binding competence when incorporated into an oligomeric complex. These findings support a model in which nucleotide binding to NBD1 is dependent on and regulated by trans−acting elements from neighboring subunits, either by direct interaction with the nucleotide or by stabilization of a nucleotide binding−competent state. In this way, they provide a basis for intersubunit communication within the functional ClpB comple

Lysine Acylation Using Conjugating Enzymes (LACE) for Site-Specific Modification and Ubiquitination of Native Proteins

Enzymes are powerful tools for post-translational protein labeling due to their high sequence specificity and mild reaction conditions. Many existing protocols, however, are restricted to conjugations at terminal positions or rely on non-peptidic metabolites and large recognition domains. Here we introduce a chemoenzymatic method to functionalize proteins at internal lysine residues that are part of genetically encoded minimal recognition tags (four residues). We achieved this by employing the intrinsic sequence specificity of the E2 SUMO-conjugating enzyme Ubc9 and a short peptide thioester, which together obviate the need for E1 and E3 enzymes. Using a range of protein substrates, we apply this approach to the conjugation of biochemical probes, one-pot dual-labeling reactions in combination with sortase, and site-specific monoubiquitination and ISG15ylation. The small tag size and large substrate tolerance of Ubc9 will make this a method of choice for protein engineering by isopeptide formation and the preparation of ubiquitinated proteins. </p

Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213