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Ethylene formation by the ethylene-forming enzyme (EFE) and 1-aminocyclopropane-1-carboxylate oxidase (ACCO).

 

The non-heme Fe( ii ) and 2-oxoglutarate (2OG) dependent ethylene-forming enzyme (EFE) catalyzes both ethylene generation and l -Arg hydroxylation. Despite experimental and computational progress in understanding the mechanism of EFE, no EFE variant has been optimized for ethylene production while simultaneously reducing the l -Arg hydroxylation activity. In this study, we show that the two l -Arg binding conformations, associated with different reactivity preferences in EFE, lead to differences in the intrinsic electric field (IntEF) of EFE. Importantly, we suggest that applying an external electric field (ExtEF) along the Fe–O bond in the EFE·Fe( iii )·OO − ˙·2OG· l -Arg complex can switch the EFE reactivity between l -Arg hydroxylation and ethylene generation. Furthermore, we explored how applying an ExtEF alters the geometry, electronic structure of the key reaction intermediates, and the individual energy contributions of second coordination sphere (SCS) residues through combined quantum mechanics/molecular mechanics (QM/MM) calculations. Experimentally generated variant forms of EFE with alanine substituted for SCS residues responsible for stabilizing the key intermediates in the two reactions of EFE led to changes in enzyme activity, thus demonstrating the key role of these residues. Overall, the results of applying an ExtEF indicate that making the IntEF of EFE less negative and stabilizing the off-line binding of 2OG is predicted to increase ethylene generation while reducing l -Arg hydroxylation. 

Many living tissues achieve functions through architected constituents with strong adhesion. An Achilles tendon, for example, transmits force, elastically and repeatedly, from a muscle to a bone through staggered alignment of stiff collagen fibrils in a soft proteoglycan matrix. The collagen fibrils align orderly and adhere to the proteoglycan strongly. However, synthesizing architected materials with strong adhesion has been challenging. Here we fabricate architected polymer networks by sequential polymerization and photolithography, and attain adherent interface by topological entanglement. We fabricate tendon-inspired hydrogels by embedding hard blocks in topological entanglement with a soft matrix. The staggered architecture and strong adhesion enable high elastic limit strain and high toughness simultaneously. This combination of attributes is commonly desired in applications, but rarely achieved in synthetic materials. We further demonstrate architected polymer networks of various geometric patterns and material combinations to show the potential for expanding the space of material properties.

 

LarC catalyzes the CTP-dependent insertion of nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide (P2TMN), the final biosynthetic step for generating the nickel-pincer nucleotide (NPN) enzyme cofactor. In this study, we characterized a LarC homolog from Moorella thermoacetica (LarCMt) and characterized selected properties of the protein. We ruled out the hypothesis that enzyme inhibition by its product pyrophosphate accounts for its apparent single-turnover activity. Most notably, we identified a cytidinylylated-substrate intermediate that is formed during the reaction of LarCMt. Selected LarCMt variants with substitutions at the predicted CTP-binding site retained substantial amounts of activity, but exhibited greatly reduced levels of the CMP-P2TMN intermediate. In contrast, enhanced amounts of the CMP-P2TMN intermediate were generated when using LarCMt from cells grown on medium without supplemental nickel. On the basis of these results, we propose a functional role for CTP in the unprecedented nickel-insertase reaction during NPN biosynthesis.

 

The LarA superfamily consists of nickeldependent enzymes catalyzing racemization/epimerization reactions using a variety of α-hydroxy acids. The first-characterized LarA, a lactate racemase from Lactobacillus plantarum, led to the discovery of the nickel-pincer nucleotide (NPN) cofactor that is utilized by family members with alternative substrates, including malate racemase from Thermoanaerobacterium thermosaccharolyticum (Mar2). In this work, a higher resolution crystal structure of Mar2 was obtained with better data quality that revealed new structural and dynamic characteristics of the protein. A model of the Mar2 structure with bound cofactor and substrate was generated to uncover the common and the unique features among two distinct subgroups in the LarA superfamily. In addition, structure-guided mutational studies were used to examine the importance of residues that are modeled to interact with NPN and to explore which residues were critical for conferring specificity for malate. In particular, substitution of two residues involved in substrate binding in Mar2 to match the corresponding residues in LarA led to the acquisition of low levels of lactate racemase activity. Of additional interest, the substrate spectrum was expanded to include tartrate, an analog of malate. These new findings will help to better understand structure–function relationships of many other LarA homologs that are broadly distributed in bacterial and archaeal species.