A Single Mutation Increases the Activity and Stability of Pectobacterium carotovorum Nitrile Reductase

Abstract

Nitrile reductases are considered to be promising and environmentally benign nitrile-reducing biocatalysts to replace traditional metal catalysts. Unfortunately, the catalytic efficiencies of the nitrile reductases reported so far are very low. To date, all attempts to increase the catalytic activity of nitrile reductases by protein engineering have failed. In this work, we successfully increased the specific activity of a nitrile reductase from Pectobacterium carotovorum from 354 to 526 U g by engineering the substrate binding pocket; moreover, the thermostability was also improved (≈2-fold), showing half-lives of 140 and 32 h at 30 and 40 °C, respectively. In the bioreduction of 2-amino-5-cyanopyrrolo[2,3-d]pyrimidin-4-one (preQ ) to 2-amino-5-aminomethylpyrrolo[2,3-d]pyrimidin-4-one (preQ ), the variant was advantageous over the wild-type enzyme with a higher reaction rate and complete conversion of the substrate within a shorter period. Homology modeling and docking analysis revealed some possible origins of the increased activity and stability. These results establish a solid basis for future engineering of nitrile reductases to increase the catalytic efficiency further, which is a prerequisite for applying these novel biocatalysts in synthetic chemistry.

Utility of B-Factors in Protein Science: Interpreting Rigidity, Flexibility, and Internal Motion and Engineering Thermostability

“…In contrast to the above programmed optimization of thermostability and stereoselectivity, cases are known in which thermostabilization also results in some degree of activity enhancement, although this appears to be a fortuitous unplanned result. Similarly, it has been observed that the programmed increase in activity of the nitrile reductase from Pectobacterium carotovorum upon exchanging amino acids within 6 Å of the bound substrate individually by alanine is accompanied by an increase in thermostability, an observation that was difficult to explain on a molecular level …”

“…Similarly, it has been observed that the programmed increase in activity of the nitrile reductase from Pectobacterium carotovorum upon exchanging amino acids within 6 Å of the bound substrate individually by alanine is accompanied by an increase in thermostability, an observation that was difficult to explain on a molecular level. 391 3.3.7. B-FIT as a Means to Increase Robustness toward Hostile Solvents.…”

Computational Insights into the Catalytic Mechanism of Bacterial Carboxylic Acid Reductase

“…Nevertheless, the natures of the transition states concerning hydride and proton transfer during the reduction step have not yet been uncovered, and the proton transfer path also remains elusive (Scheme ). In contrast, the reduction mechanisms of many other NADPH-dependent reductases, including NRPSs, , short-chain dehydrogenases, − cinnamoyl-CoA reductases, fatty acyl-CoA reductases, HMG-CoA reductase, , and nitrile reductases, , have already been elucidated. In fact, the CAR R domain may adopt the SDR reduction mechanism (Scheme S1a), as it strongly resembles the reductase domain of the SDR family on the structural level .…”

Engineered ketoreductase-catalyzed stereoselective reduction of ethyl 2′-ketopantothenate and its analogues: chemoenzymatic synthesis of d-pantothenic acid

“…On the other hand, we included residues M242 and P243 in our mutagenesis campaign, as mutation of these two residues was previously reported to provide SSCR variants with improved catalytic properties in the asymmetric reduction of other ketone substrates. 28,29 With 15 candidate amino acid residues in hand, we decided to perform alanine scanning first, 30,31 prior to conducting more laborintensive and time-consuming saturation mutagenesis. Accordingly, site-directed mutagenesis was used to construct the required alanine mutants.…”