Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

Bjelic, Sinisa; Kipnis, Yakov; Pianowski, Zbigniew; Vorobiev, S.M.; Su, Min; Seetharaman, J.; Xiao, Rong; Kornhaber, G.; Hunt, J.F.; Tong, Liang; Hilvert, Donald; Baker, David

doi:10.1016/j.jmb.2013.10.012

Cited by 36 publications

(45 citation statements)

References 43 publications

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“…Thus, it can be incorporated into cells to assess proteostasis network influences on RA folding without concerns about loss-or gain-of-function cellular phenotypes. The RA of focus, RA114.3 (14), adopts an eight-stranded α/β barrel fold (Fig. S1A), a common natural enzyme scaffold (15).…”

Section: Resultsmentioning

confidence: 99%

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

Liu

Tan

Zhang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein's folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/ chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein's functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.chemical probes | pharmacologic chaperone | fluorescence labeling A ll proteins are biosynthesized as linear chains, and most need to fold into 3D structures to function. Studies on protein folding in buffers have revealed that a kinetic competition typically exists between protein folding, misfolding, and aggregation. It is the role of the protein homeostasis or proteostasis network in each subcellular compartment to regulate this competition and keep the folded and functional proteome within the physiological concentration range, while minimizing misfolding and aggregation in the face of stresses (1-4). It remains a challenge to discern how the proteostasis network affects the folding of proteins into biologically active conformations required for function in vivo (5).Current methodologies allow for quantification of the partitioning of a protein of interest (POI) between soluble and aggregated states but cannot determine the proportion of the soluble population that is properly folded and functional. Published folding probes have the potential to report on the folded fraction in cells or cell lysate (6-9); however, the extent to which they shift folding equilibria and quantify the folded and functional fraction faithfully has not been studied. Herein, we create POI folding probes by adapting the principle of activity-based protein profiling (10) to quantify the soluble folded and functional fraction of a particular protein in a cell lysate. We seek folding probes that bind to and...

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Section: Resultsmentioning

confidence: 99%

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

Liu

Tan

Zhang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…[98,95] Cassette mutagenesis of active-site residues, followed by multiple rounds of error-prone polymerase chain reaction (PCR) mutagenesis and gene shuffling, afforded >1000-fold enhancements of the starting activities. [100] The most evolved retro-aldolases exhibited rate accelerations >10 8 -fold over the simple lysine-catalyzed reaction and, notably, had k cat values that are similar to or even exceed those of the best aldolase antibodies. Furthermore, their k cat /K M values approached 10 3 M -1 • s -1 , which is within a factor of 100 of the k cat /K M values for natural aldolases.…”

Section: 1123mentioning

confidence: 94%

3.11 Creation and Optimization of Artificial Enzymes for Abiological Reactions

Faber¹,

Fessner²,

Turner³

2015

Biocatalysis in Organic Synthesis 3

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Enzymes, by far the most abundant and versatile catalysts in nature, are of enormous interest to organic chemists. High rate accelerations and exacting selectivities under mild, environmentally friendly conditions make them useful, alone or in combination with other enzymes, for the sustainable production of fine chemicals, [1][2][3][4] bioremediation, [5,6] medicine, [7,8] and diagnostic technologies. [9] Nevertheless, natural enzymes are seldom optimal for nonphysiological applications. Low stability, cosolvent sensitivity, product inhibition, and restricted substrate and reaction scope often limit their practical utility. [10] Advances in molecular biology and protein crystallography have aided efforts to finetune enzyme properties for new applications. Because it is not always possible to predict the precise outcome of protein mutation, however, evolutionary algorithms are increasingly utilized to guide enzyme optimization by a process akin to Darwinian evolution. [11][12][13] By facilitating the identification and recombination of favorable amino acid substitutions, "directed evolution" is useful for altering regio-or stereochemical outcomes, [14][15][16] enabling recognition of unnatural substrates, [17] boosting minor promiscuous activities, [18][19][20] and improving tolerance to aggressive environments. [10] Like its natural counterpart, directed evolution is comprised of three steps: creation of diversity, selection of desired phenotypes, and amplification of the selected variants. These steps can be performed iteratively, in a "steepest descent"-like optimization process, for any screenable or selectable property; no foreknowledge of structure or mechanism is required. Over the last two decades, robust methods for sequence diversification have been developed, including error-prone polymerase chain reaction (PCR) mutagenesis, [21] targeted cassette mutagenesis, [22] and DNA shuffling. [23] Coupled with powerful screening assays and selection systems, these techniques can be reliably applied to the optimization of enzymes for academic, medical, and industrial purposes. Other mutagenesis strategies, such as neutral drift [24,25] or "substrate walking", [26] have proven useful for achieving larger changes in activity or specificity. As a consequence, it is now routinely possible to adapt enzymes to a specific process of interest rather than vice versa. [2] Despite numerous examples of successful enzyme engineering, dramatic changes in function are rare and generally serendipitous. As an alternative to tweaking existing activities, de novo enzyme design is a potentially more effective strategy for producing new catalysts for completely arbitrary chemical transformations. A variety of experimental approaches have been explored toward this end. Presently, catalytic antibody technology and computational enzyme design are among the most successful (Figure 1; see Section 3.11.1). Although their impact on biocatalysis is still limited, these methods point the way for the future. This review highlights the advanta...

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“…Retro‐aldolases that exploit amine catalysis are the mechanistically most complex enzymes designed to date 2b. 3a, 4 They utilize a reactive active‐site lysine to promote a multistep reaction sequence involving several enzyme‐bound Schiff base intermediates. Following laboratory evolution, activities approaching those of natural class I aldolases have been attained 3b.…”

Section: Methodsmentioning

confidence: 99%

A Promiscuous De Novo Retro‐Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates

2015

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Recent advances in computational design have enabled the development of primitive enzymes for ar ange of mechanistically distinct reactions.H ere we show that the rudimentary active sites of these catalysts can give rise to useful chemical promiscuity.S pecifically,R A95.5-8, designed and evolved as ar etro-aldolase,a lso promotes asymmetric Michael additions of carbanions to unsaturated ketones with high rates and selectivities.T he reactions proceed by amine catalysis,a si ndicated by mutagenesis and X-ray data. The inherent flexibility and tunability of this catalyst should make it av ersatile platform for further optimization and/or mechanistic diversification by directed evolution.

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Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

Cited by 36 publications

References 43 publications

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

3.11 Creation and Optimization of Artificial Enzymes for Abiological Reactions

A Promiscuous De Novo Retro‐Aldolase Catalyzes Asymmetric Michael Additions via Schiff Base Intermediates

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