The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10 5 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a .200-fold increase in k cat /K m (k cat /K m of 2,600 M 21 s 21 and k cat /k uncat of .10 6 ). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.Naturally occurring enzymes are extraordinarily efficient catalysts 1 . They bind their substrates in a well-defined active site with precisely aligned catalytic residues to form highly active and selective catalysts for a wide range of chemical reactions under mild conditions. Nevertheless, many important synthetic reactions lack a naturally occurring enzymatic counterpart. Hence, the design of stable enzymes with new catalytic activities is of great practical interest, with potential applications in biotechnology, biomedicine and industrial processes. Furthermore, the computational design of new enzymes provides a stringent test of our understanding of how naturally occurring enzymes work. In the past several years, there has been exciting progress in designing new biocatalysts 2,3 .Here we describe the use of our recently developed computational enzyme design methodology 4 to create new enzyme catalysts for a reaction for which no naturally occurring enzyme exists: the Kemp elimination 5,6 . The reaction, shown in Fig. 1a, has been extensively studied as an activated model system for understanding the catalysis of proton abstraction from carbon-a process that is normally restricted by high activation-energy barriers 7,8 . Computational design methodThe first step in our protocol for designing new enzymes is to choose a catalytic mechanism and then to use quantum mechanical transition state calculations to create an idealized active site with protein functional groups positioned so as to maximize transition state stabilization (Fig. 1b). The key step for the Kemp elimination is deprotonation of a carbon by a general base. We chose two different catalytic bases for this purpose: first, the carboxyl group of an aspartate or glutamate side chain, and, second, the imidazole of a histidine positioned and polarized by the carboxyl group of an aspartate or glutamate (we refer to this combination as a His-Asp dyad). The two choices have complementary strengths and weaknesses. The advantage of the carboxylate...

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.It is generally accepted that the three-dimensional structure of a polypeptide chain is determined by its amino acid sequence. Nevertheless, similar folds can have very different sequences. One of the ultimate goals in sequence analysis is to predict the structure and function of a protein based solely on its sequence. In cases where the protein of interest shares at least 30% amino acid identity with another protein, the two proteins generally exhibit similar three-dimensional structure (Doolittle 1986). Alternatively, when proteins are known to have similar structure but divergent sequences, consensus sequence motifs can be used to assess the function of unassigned sequences. These consensus motifs usually correspond to residues interacting with cofactors, substrate, or other proteins.The increasing number of three-dimensional structures of proteins in the Protein Data Bank, complexed with appropriate ligands, provides an important tool for understanding the mechanisms of molecular recognition. In this study, we focussed on flavin adenine dinucleotide (FAD) because it and its related cofactors, nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP), appear in many biological processes.Previous comparative structural studies of mononucleotide-and dinucleot...

SummaryUpon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at ∼2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20°C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.

Humans can be infected by SARS-CoV-2 either through inhalation of airborne viral particles or by touching contaminated surfaces. Structural and functional studies have shown that a single RBD of the SARS-CoV-2 homotrimer spike glycoprotein interacts with ACE2, which serves as its receptor 1,2 . Binding of spike (S) protein to ACE2 and subsequent cleavage by the host protease transmembrane serine protease 2 (TMPRSS2) results in cell and virus membrane fusion and cell entry 1 . Blocking of the ACE2 receptor by specific antibodies prevents viral entry 1,3-5 . In vitro binding measurements have shown that SARS-CoV-2 S protein binds ACE2 with an affinity of around 10 nM, which is about tenfold tighter than the binding of the SARS-CoV S protein 2,4,6 . It has been suggested that this is, at least partially, responsible for the higher infectivity of SARS-CoV-2 7 . Recently, three major SARS-CoV2 variants of concern have emerged and mutations in the RBD of the spike proteins of these variants have further strengthened this hypothesis. Deep-mutational scanning of the RBD domain showed that the N501Y mutation in the Alpha variant to enhances binding to ACE2 7 . The Beta variant has three altered residues in the ACE2-binding site (K417N, E484K and N501Y), and has spread extremely rapidly, becoming the dominant lineage in the Eastern Cape and Western Cape Provinces within weeks 8 . The Gamma variant, with independent K417T, E484K and N501Y mutations, similar to the B.1.351 variant is spreading rapidly from the Amazon region 9 . Another S mutation associated with increased SARS-CoV-2 infectivity is S477N, which became dominant in many regions 10 .Efficacious vaccines are now being administered 11 . However, especially when a large fraction of the global population remains unvaccinated, the potential of the continuously mutating virus to become at least partially resistant to vaccines means that drug development must continue. Potential therapeutic targets that block viral entry include molecules that block the spike protein, the TMPRSS2 protease or the ACE2 receptor 12 . Multiple high-affinity neutralizing antibodies have been developed 13 . Soluble forms of the ACE2 protein 14,15 or engineered parts or mimics have also shown efficacy 16,17 . In addition, previously developed TMPRSS2 inhibitors have been repurposed for treatment of COVID-19 1 .The development of molecules to block the ACE2 protein has not received much attention. One potential caveat with this approach is the importance of ACE2 biological activity, both as a carboxypeptidase removing a single C-terminal amino acid from angiotensin II to generate angiotensin-(1-7) and in the regulation of amino acid transport and pancreatic insulin secretion 18,19 . Dalbavancin is a drug that blocks the spike protein-ACE2 interaction, however it does so with low affinity 20 (approximately 130 nM).We hypothesized that the RBD domain of SARS-CoV-2 could be used as a competitive inhibitor of the ACE2 receptor binding site. However, this would probably require an RBD with picomola...

In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.