Biology subjects

Gutierrez-Rus, L. I.

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

3 recordsLinked to original sources

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↗