The road to fully programmable protein catalysis

Lovelock, Sarah L.; Crawshaw, Rebecca; Basler, Sophie; Levy, Colin; Baker, David; Hilvert, Donald; Green, Anthony P.

doi:10.1038/s41586-022-04456-z

Cited by 215 publications

(153 citation statements)

References 114 publications

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“…Recent single-molecule experiments 49,[59][60][61][62][63] have generally shown two active states of APs from several different origins. Earlier studies using capillary electrophoresis to see product formation by individual AP molecules indicated up to ten differently active kinetic states, with three of the active forms being dominating 59 .…”

Section: Discussionmentioning

confidence: 99%

Structural characterization of functionally important chloride binding sites in the marine Vibrio alkaline phosphatase

Markússon

Hjörleifsson

Kursula

et al. 2022

Preprint

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Enzyme stability and function can be affected by various environmental factors, such as temperature, pH and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0 – 1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small angle X-ray scattering (SAXS) showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.

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Section: Discussionmentioning

confidence: 99%

Structural characterization of functionally important chloride binding sites in the marine Vibrio alkaline phosphatase

Markússon

Hjörleifsson

Kursula

et al. 2022

Preprint

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“…The generation of de novo enzymes ( i.e ., enzymes with completely new active sites) is one of the major unsolved problems in protein engineering [32]. Besides the obvious biotechnological implications, the results of de novo enzyme engineering studies may also have immediate implications for our understanding of the origin of life.…”

Section: Discussionmentioning

confidence: 99%

“…However, such efficient mechanisms are not apparent in the efforts of protein engineers to develop completely new enzyme functionalities. Only a limited number of de novo enzymes have been reported to date [32]and several of the most recent success stories in this field involve the recruitment of metals or metal-containing cofactors, which already provide by themselves some starting level of catalysis. However, only about 30% of enzymes are metalloenzymes [43,44]and mechanisms for the emergence of new enzymes that do not rely on metal recruitment remain poorly understood.…”

Section: Discussionmentioning

confidence: 99%

Efficient base-catalysed Kemp elimination in an engineered ancestral enzyme

Gutierrez-Rus

Alcalde

Risso

et al. 2022

Preprint

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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=2x105 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.

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“…20 In both studies, extensive screening of single-site mutation libraries and their combination provided highly active enzyme variants over four to five rounds of evolution. While it is unclear if similar engineering campaigns could expand the promiscuity of nucleoside phosphorylases to provide true "generalists", we expect that recent advances in sequencing technology 56 and the advent of reliable computational methods for enzyme design 57 will accelerate the expansion of the substrate scope of these enzymes.…”

Section: New Approaches For Efficient Enzyme Preparationmentioning

confidence: 99%

Industrial potential of the enzymatic synthesis of nucleoside analogs: Existing challenges and perspectives

Westarp

Kaspar

Neubauer

et al. 2022

Preprint

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Nucleoside phosphorylases have progressed from an enzymatic curiosity to a viable synthetic tool. However, despite the recent advances in nucleoside phosphorylase-catalyzed nucleoside synthesis, the widespread application of these enzymes in industrial processes is still lacking. We attribute this gap to three key challenges, which are outlined in this short review. To address these persistent obstacles, we believe that biocatalytic nucleoside synthesis needs to embrace interdisciplinary partnerships with the fields of organic chemistry, process engineering and flow chemistry.

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The road to fully programmable protein catalysis

Cited by 215 publications

References 114 publications

Structural characterization of functionally important chloride binding sites in the marine Vibrio alkaline phosphatase

Structural characterization of functionally important chloride binding sites in the marine Vibrio alkaline phosphatase

Efficient base-catalysed Kemp elimination in an engineered ancestral enzyme

Industrial potential of the enzymatic synthesis of nucleoside analogs: Existing challenges and perspectives

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