Progress, Challenges, and Opportunities with Artificial Metalloenzymes in Biosynthesis

Bloomer, Brandon J.; Clark, Douglas S.; Hartwig, John F.

doi:10.1021/acs.biochem.1c00829

Cited by 25 publications

(21 citation statements)

References 44 publications

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“…Specifically, we targeted the periplasmic space of Escherichia coli (E. coli) cells, which is more permissive in terms of metal/chemical exchange with the external medium compared to the cytosol and thus has proven to be particularly suitable for the development of artificial metalloproteins/metalloenzymes with in vivo functions. ,,− Conveniently, cytochrome cb 562 (parent of B 2 ) is equipped with an N -terminal signal sequence for Sec-dependent translocation into the periplasmic space, whose oxidative environment enables disulfide bond formation for B 2 dimer assembly. In our experiments, B 2 was expressed in E.…”

Section: Resultsmentioning

confidence: 99%

Design of a Flexible, Zn-Selective Protein Scaffold that Displays Anti-Irving–Williams Behavior

Choi

Tezcan

2022

J. Am. Chem. Soc.

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Selective metal binding is a key requirement not only for the functions of natural metalloproteins but also for the potential applications of artificial metalloproteins in heterogeneous environments such as cells and environmental samples. The selection of transition-metal ions through protein design can, in principle, be achieved through the appropriate choice and the precise positioning of amino acids that comprise the primary metal coordination sphere. However, this task is made difficult by the intrinsic flexibility of proteins and the fact that protein design approaches generally lack the sub-Å precision required for the steric selection of metal ions. We recently introduced a flexible/ probabilistic protein design strategy (MASCoT) that allows metal ions to search for optimal coordination geometry within a flexible, yet covalently constrained dimer interface. In an earlier proof-of-principle study, we used MASCoT to generate an artificial metalloprotein dimer, (AB) 2 , which selectively bound Co II and Ni II over Cu II (as well as other first-row transition-metal ions) through the imposition of a rigid octahedral coordination geometry, thus countering the Irving−Williams trend. In this study, we set out to redesign (AB) 2 to examine the applicability of MASCoT to the selective binding of other metal ions. We report here the design and characterization of a new flexible protein dimer, B 2 , which displays Zn II selectivity over all other tested metal ions including Cu II both in vitro and in cellulo. Selective, anti-Irving−Williams Zn II binding by B 2 is achieved through the formation of a unique trinuclear Zn coordination motif in which His and Glu residues are rigidly placed in a tetrahedral geometry. These results highlight the utility of protein flexibility in the design and discovery of selective binding motifs.

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Section: Resultsmentioning

confidence: 99%

Design of a Flexible, Zn-Selective Protein Scaffold that Displays Anti-Irving–Williams Behavior

Choi

Tezcan

2022

J. Am. Chem. Soc.

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“…In comparison with WT‐SPS which is our mimicking target enzyme to produce styrylpyrone ( 1 ) in nature, The k cat of 3AP‐L261G‐2PS was over 58‐fold greater than that of WT‐SPS (Table 3) and the overall catalytic efficiency ( k cat / K M ) was improved by 30 folds. More importantly, the catalytic efficiency of the engineered 3AP‐L261G‐2PS is even higher than those of many native type III polyketide synthases’ reactivity (3600 s −1 M −1 for WT CHS and 4200 s −1 M −1 for WT STS) that have been frequently applied in metabolic engineering researches [35,39,40] . This provided a highly promising platform for higher level metabolic engineering where 2PS mutant could potentially produce styrylpyrone products naturally generated by SPS.…”

Section: Resultsmentioning

confidence: 99%

“…More importantly, the catalytic efficiency of the engineered 3AP-L261G-2PS is even higher than those of many native type III polyketide synthases' reactivity (3600 s À 1 M À 1 for WT CHS and 4200 s À 1 M À 1 for WT STS) that have been frequently applied in metabolic engineering researches. [35,39,40] This provided a highly promising platform for higher level metabolic engineering where 2PS mutant could potentially produce styrylpyrone products naturally generated by SPS. Further kinetic experiments have been conducted on other 3AP mutants (Table 3) and the increased sterics at 261 position not only decreased the transformation yield, but also lowered the turnover number and catalytic efficiency (Supplementary Figure S5).…”

Section: Condition Optimization and Kineticsmentioning

confidence: 99%

Reshaping the 2‐Pyrone Synthase Active Site for Chemoselective Biosynthesis of Polyketides

Mirts

Yook

et al. 2022

Angewandte Chemie

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Engineering enzymes with novel reactivity and applying them in metabolic pathways to produce valuable products are quite challenging due to the intrinsic complexity of metabolic networks and the need for high in vivo catalytic efficiency. Triacetic acid lactone (TAL), naturally generated by 2‐pyrone synthase (2PS), is a platform molecule that can be produced via microbial fermentation and further converted into value‐added products. However, these conversions require extra synthetic steps under harsh conditions. We herein report a biocatalytic system for direct generation of TAL derivatives under mild conditions with controlled chemoselectivity by rationally engineering the 2PS active site and then rewiring the biocatalytic pathway in the metabolic network of E. coli to produce high‐value products, such as kavalactone precursors, with yields up to 17 mg/L culture. Computer modeling indicates sterics and hydrogen‐bond interactions play key roles in tuning the selectivity, efficiency and yield.

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“…Current and planned applied work in progress include the application of LMA’s unique features to declutter vibrational spectra, such as assisting isotope-edited IR spectroscopy pinpointing specific bonds of interest in the IR spectrum of DNA, which is hampered by the large number of overlapping carbonyl signals; monitoring oxidation state changes in photoactive enzymes via changes in LMA properties in support of, e.g., protein film electrochemistry; combining LMA with the group’s unified reaction valley approach (URVA) to model properties of artificial metalloenzymes and their catalytic mechanisms; , applying LMA’s potential in the emerging field of covalent binder drugs, which strongly depends on reliable, quantitative bond strength descriptors; , and on the other end of the spectrum, investigating a large number of reported ionic crystals with the far-reaching goal to compile a database with individual local mode force constants, serving as a basis for comprehensive studies on how important physicochemical properties, e.g., electrical conductivity and optical properties of these crystals are related to the individual bond strengths in these materials.…”

Section: Summary and Concluding Remarksmentioning

confidence: 99%

The Local Vibrational Mode Theory and Its Place in the Vibrational Spectroscopy Arena

et al. 2022

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This Feature Article starts highlighting some recent experimental and theoretical advances in the field of IR and Raman spectroscopy, giving a taste of the breadth and dynamics of this striving field. The local mode theory is then reviewed, showing how local vibrational modes are derived from fundamental normal modes. New features are introduced that add to current theoretical efforts: (i) a unique measure of bond strength based on local mode force constants ranging from bonding in single molecules in different environments to bonding in periodic systems and crystals and (ii) a new way to interpret vibrational spectra by pinpointing and probing interactions between particular bond stretching contributions to the normal modes. All of this represents a means to work around the very nature of normal modes, namely that the vibrational motions in polyatomic molecules are delocalized. Three current focus points of the local mode analysis are reported, demonstrating how the local mode analysis extracts important information hidden in vibrational spectroscopy data supporting current experiments: (i) metal−ligand bonding in heme proteins, such as myoglobin and neuroglobin; (ii) disentanglement of DNA normal modes; and (iii) hydrogen bonding in water clusters and ice. Finally, the use of the local mode analysis by other research groups is summarized. Our vision is that in the future local mode analysis will be routinely applied by the community and that this Feature Article serves as an incubator for future collaborations between experiment and theory.

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Progress, Challenges, and Opportunities with Artificial Metalloenzymes in Biosynthesis

Cited by 25 publications

References 44 publications

Design of a Flexible, Zn-Selective Protein Scaffold that Displays Anti-Irving–Williams Behavior

Design of a Flexible, Zn-Selective Protein Scaffold that Displays Anti-Irving–Williams Behavior

Reshaping the 2‐Pyrone Synthase Active Site for Chemoselective Biosynthesis of Polyketides

The Local Vibrational Mode Theory and Its Place in the Vibrational Spectroscopy Arena

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