Biology subjects

Gutierrez-Rus, L. I.

Publications and source records attributed to Gutierrez-Rus, L. I..

5 recordsLinked to original sources

bioRxiv2026

Expanding all-α-helical protein space through rational computational design

De novo protein design is advancing rapidly1,2. This is being driven by AI to generate protein backbones, sequences, and structural models3-7. As a result, de novo designed proteins are becoming larger and more complex8-10, and increasingly explore new protein structures11,12. By contrast, natural proteins have evolved structural and functional complexity by modular combination of recurring protein domains13. Approximately 25% of these natural domains are mostly -helical structures14. Here we show how these can be expanded using rational computational design. Following the domain classification scheme CATH15, we build complex all- de novo proteins hierarchically using sequence-to-structure relationships for helix-helix interactions, systematic rules to connect helices, computational tools to design loops, and in silico evaluation. The pipeline starts with a target architecture of free-standing helices. These are connected into a topology by considering local arrangements of helical bundles using understood sequence-to-structure relationships for helix packing. Single-chain sequences are completed using template- and AI-based methods. Finally, AlphaFold models are assessed to give small numbers of designs for experimental validation. We test 31 designs for 14 different architectures and 25 topologies. 75% of these express as stable, monomeric, water-soluble proteins; and >30% yield X-ray crystal structures matching the designs to atomic accuracy and with new-to-nature structures. Finally, several of the scaffolds are functionalised through one-shot designs to deliver ion, small-molecule and protein binders.

synthetic biology↗

bioRxiv2024

Ancestral versus Modern Substrate Scope in Family-1 Glycosidases

Experimental studies support that protein engineering based on ancestral sequence reconstruction often leads to variants with biotechnologically useful biomolecular properties. These may include high stability, enhanced conformational flexibility and a modified catalysis range. Carbohydrate-active enzymes have numerous applications related with the degradation and synthesis of carbohydrates and glycoconjugates. Here, we explore how ancestral reconstruction may impact substrate scope in glycosidases, highly diverse enzymes that catalyze the hydrolysis of glycosidic bonds in all living cells and find applications as catalysts of the reverse reaction. To this end, we screen a library of [~]500 potential glycosidase substrates for degradation by both, a modern family-1 glycosidase from Halothermothrix orenii and a putative ancestral family-1 glycosidase derived from sequence reconstruction at a bacterial-eukaryotic common ancestor. The modern enzyme is the better catalyst for most substrates. But the ancestral glycosidase is more efficient with flavonoid glycosides bearing large aglycon moieties. Analysis of the catalytic parameters for a selected set of substrates, alongside analysis of the library data using a supervised learning algorithm, support the hypothesis that the modern enzyme tends to become less catalytically efficient with increasing substrate size, while this trend is not observed for the ancestral glycosidase. Molecular simulations support that the ancestral catalysis pattern is linked to the existence of a highly flexible region of the ancestral structure and a cavity capable of accommodating large aglycons. Our results provide guidelines for the engineering of enzymes for the synthesis and hydrolysis of large glycoconjugates.

biochemistry↗

bioRxiv2023

Protection of catalytic cofactors by polypeptides as a driver for the emergence of primordial enzymes

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects protection of bound heme from common degradation processes and a resulting longer life time and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

molecular biology↗

bioRxiv2022

Efficient base-catalysed Kemp elimination in an engineered ancestral enzyme

The routine generation of enzymes with completely new active sites is one of the major unsolved problems in protein engineering. Advances in this field have been so far modest, perhaps due, at least in part, to the widespread use of modern natural proteins as scaffolds for de novo engineering. Most modern proteins are highly evolved and specialized, and, consequently, difficult to repurpose for completely new functionalities. Conceivably, resurrected ancestral proteins with the biophysical properties that promote evolvability, such as high stability and conformational diversity, could provide better scaffolds for de novo enzyme generation. Kemp elimination, a non-natural reaction that provides a simple model of proton abstraction from carbon, has been extensively used as a benchmark in de novo enzyme engineering. Here, we present an engineered ancestral {beta}-lactamase with a new active site capable of efficiently catalysing the Kemp elimination. Our Kemp eliminase is the outcome of a minimalist design based on a single function-generating mutation followed by sharply-focused, low-throughput library screening. Yet, its catalytic parameters (kcat/KM=2{middle dot}105 M-1s-1, kcat=635 s-1) compare favourably with the average modern natural enzyme and with the best proton-abstraction de novo Kemp eliminases reported in the literature. General implications of our results for de novo enzyme engineering are discussed.

biophysics↗

bioRxiv2022

Cell survival enabled by leakage of a labile metabolic intermediate

Many metabolic pathways are of ancient origin and have evolved over long periods of time (Noda-Garcia et al., 2018). Yet, new pathways can also emerge in short time scales in response, for instance, to the presence of anthropogenic chemicals in the environment (Copley, 2009). Models of metabolic pathway emergence and evolution often emphasize the acquisition of new reactions through horizontal gene transfer and promiscuous enzyme functionalities (Pal et al., 2005; Schulenburg & Miller, 2014; Copley, 2015; Noda-Garcia et al., 2018; Peracchi, 2018). A fundamentally different mechanism of metabolic innovation is revealed by the evolutionary repair experiments reported here. A block in the proline biosynthetic pathway that compromises cell survival is efficiently rescued by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a sequestered phosphorylated intermediate common to the biosynthetic pathways of proline and glutamine, thus generating a new route to proline. Metabolic intermediates may undergo a variety of chemical and enzymatic transformations, but are typically protected as shielded reaction intermediates or through channeling in multi-enzyme complexes and metabolons (Srere, 1987; Huang et al., 2001; Grunwald, 2018; Pareek et al., 2021). Our results show that intermediate leakage can readily occur and contribute to organismal adaptation. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways. We therefore anticipate applications of mutation-induced leakage in metabolic engineering.

molecular biology↗