Not satisfied with biology’s vast catalyst repertoire, I want to create new enzyme catalysts and expand the chemistry of life. We use the most powerful biological design process, evolution, to optimize existing enzymes and invent new ones, thereby circumventing our profound ignorance of how sequence encodes function. As beautifully demonstrated by Dan Tawfik, evolution not only optimizes enzyme function, it also innovates by exploiting promiscuous catalytic activities of extant proteins to mold new enzymes. We use this insight to explore the future. I will describe how whole new families of carbene and nitrene transferase enzymes have been generated by exploiting the promiscuous capabilities of heme proteins. These new-to-nature biocatalysts increase the scope of genetically encoded chemistry.
Enzymes are extraordinary catalysts that can accelerate chemical reactions by many orders of magnitude. Yet, how optimized are enzyme catalysts? This question is addressed in two remarkable pieces from 2011 and 2015 titled “the moderately efficient enzyme” by Dan Tawfik and co-workers. They show that, on average, only 1 in 10,000 encounters between substrate molecules and active sites were productive. During my tenure in Dan’s laboratory, we explored some aspects of this ‘moderate’ efficiency. Specifically, we investigated enzymatic temperature dependencies, and found that all enzymes, independent of their adaption to extreme temperature, exhibit similar temperature dependencies and can not track the rate increase of corresponding spontaneous rates. A possible reason for the enzymes ‘moderate’ efficiency is their relative ‘floppiness’, as they exist as an ensemble of conformations. This feature may also be the origin of promiscuous activity, an important mechanism in the emergence of new enzymatic functions. To illustrate this phenomenon, I will review the case of phosphotriesterases and their progenitor lactonases that provide mechanistic insights on how different reactions can occur within the same active site. These observations pose the question: how optimized proteins can be? We focused on the bacterial phosphate transporter, including representatives from organisms living in arsenate-rich environments. While phosphate is essential and arsenate is toxic, both anions are chemically nearly identical and their discrimination is challenging. We could show that the phosphate transporter discriminates between these two anions, and more so in organisms living in high arsenate conditions. The transporter achieves this prowess via a unique, extremely finely tuned, low-barrier hydrogen bond that allows the transporter and thereby microbes to prefer phosphate over arsenate by ~1,000-fold. Further characterizations of the system reveal conditions for these bonds to form, suggesting their potential existence and putative importance in other optimized systems.
Proteins mediate the critical processes of life and beautifully solve the challenges faced during the evolution of modern organisms. Our goal is to design a new generation of proteins that address current-day problems not faced during evolution. In contrast to traditional protein engineering efforts, which have focused on modifying naturally occurring proteins, we design new proteins from scratch based on Anfinsen’s principle that proteins fold to their global free energy minimum. We compute amino acid sequences predicted to fold into proteins with new structures and functions, produce synthetic genes encoding these sequences, and characterize them experimentally. In this talk, I will describe the de novo design of SARS-CoV-2 candidate therapeutics, synthetic antagonists and agonists of cellular receptors, molecular machines, and recent advances in deep learning-based structure modeling and design.
Although bespoke, sequence-specific proteases have the potential to advance biotechnology and medicine, generation of proteases with tailor-made cleavage specificities remains a major challenge. We developed a phage-assisted protease evolution system with simultaneous positive and negative selection, and applied it to three botulinum neurotoxin (BoNT) light-chain proteases. We evolved BoNT/X protease into separate variants that preferentially cleave vesicle-associated membrane protein 4 (VAMP4) and Ykt6, evolved BoNT/F protease to selectively cleave the non-native substrate VAMP7, and evolved BoNT/E protease to cleave phosphatase and tensin homolog (PTEN) but not any natural BoNT protease substrate in neurons. The evolved proteases display large changes in specificity (218- to >11,000,000-fold) and can retain their ability to form holotoxins that self-deliver into primary neurons. These findings establish a versatile platform for reprogramming proteases to selectively cleave new targets of therapeutic interest.
mRNA decay is critical to post-transcriptional regulation of gene expression. Removal of the 5′ 7-methylguanosine (m7G) cap is the first step of 5′-3′ mRNA decay. Three NUDIX hydrolases involved in canonical m7G-cap decapping have been identified in metazoan, among which the DCP2 protein is considered as the major decapping enzyme. DCP2 functions as part of a large macromolecular decapping complex in association with regulatory proteins that conformationally gate its activity. We previously showed that DCP2 targets nearly 2000 cellular mRNAs, a majority of which are associated with P-bodies, phase-separated liquid granules enriched for 5′-3′ mRNA decay factors. However, the mechanism of DCP2 specificity remains incompletely answered. Here, we report a human microprotein called NBDY as a regulator of DCP2 specificity. NBDY localizes to P-bodies via direct interaction with the human decapping complex components EDC4 and DCP1A. Global profiling of RNA stability changes during NBDY depletion reveals more than 1400 transcripts regulated by NBDY. Particularly, fate of DCP2 substrates in NBDY knockout cells correlates with the length of 5′ untranslated region (UTR). Non-DCP2 target transcripts are stabilized by NBDY deletion as well, possibly as a result of dysregulation of alternative mRNA decay pathways. Together, we present a comprehensive model of the regulation of mRNA decapping by NBDY.