Alpha Carbonic Anhydrase 5 Mediates Stimulation of ATP Synthesis by Bicarbonate in Isolated Arabidopsis Thylakoids.

We studied bicarbonate-induced stimulation of photophosphorylation in thylakoids isolated from leaves of Arabidopsis thaliana plants. This stimulation was not observed in thylakoids of wild-type in the presence of mafenide, a soluble carbonic anhydrase inhibitor, and was absent in thylakoids of two mutant lines lacking the gene encoding alpha carbonic anhydrase 5 (αCA5). Using mass spectrometry, we revealed the presence of αCA5 in stromal thylakoid membranes of wild-type plants. A possible mechanism of the photophosphorylation stimulation by bicarbonate that involves αCA5 is proposed.

Advances in understanding the physiological role and locations of carbonic anhydrases in C3 plant cells.

The review describes the structures of plant carbonic anhydrases (CAs), enzymes catalyzing the interconversion of inorganic carbon forms and belonging to different families, as well as the interaction of inhibitors and activators of CA activity with the active sites of CAs in representatives of these families. We outline the data that shed light on the location of CAs in green cells of C3 plants, algae and angiosperms, with the emphasis on the recently obtained data. The proven and proposed functions of CAs in these organisms are listed. The possibility of the involvement of several chloroplast CAs in acceleration of the conversion of bicarbonate to CO2 and in supply of CO2 for fixation by Rubisco is particularly considered. Special attention is paid to CAs in various parts of thylakoids and to discussion about current knowledge of their possible physiological roles. The review states that, despite the significant progress in application of the mutants with suppressed CAs synthesis, the approach based on the use of the inhibitors of CA activity in some cases remains quite effective. Combination of these two approaches, namely determining the effect of CA activity inhibitors in plants with certain knocked-out CA genes, turns out to be very useful for understanding the functions of other CAs.

One-Pot Biocatalytic Transformation of Adipic Acid to 6-Aminocaproic Acid and 1,6-Hexamethylenediamine Using Carboxylic Acid Reductases and Transaminases.

Production of platform chemicals from renewable feedstocks is becoming increasingly important due to concerns on environmental contamination, climate change, and depletion of fossil fuels. Adipic acid (AA), 6-aminocaproic acid (6-ACA) and 1,6-hexamethylenediamine (HMD) are key precursors for nylon synthesis, which are currently produced primarily from petroleum-based feedstocks. In recent years, the biosynthesis of adipic acid from renewable feedstocks has been demonstrated using both bacterial and yeast cells. Here we report the biocatalytic conversion/transformation of AA to 6-ACA and HMD by carboxylic acid reductases (CARs) and transaminases (TAs), which involves two rounds (cascades) of reduction/amination reactions (AA → 6-ACA → HMD). Using purified wild type CARs and TAs supplemented with cofactor regenerating systems for ATP, NADPH, and amine donor, we established a one-pot enzyme cascade catalyzing up to 95% conversion of AA to 6-ACA. To increase the cascade activity for the transformation of 6-ACA to HMD, we determined the crystal structure of the CAR substrate-binding domain in complex with AMP and succinate and engineered three mutant CARs with enhanced activity against 6-ACA. In combination with TAs, the CAR L342E protein showed 50-75% conversion of 6-ACA to HMD. For the transformation of AA to HMD (via 6-ACA), the wild type CAR was combined with the L342E variant and two different TAs resulting in up to 30% conversion to HMD and 70% to 6-ACA. Our results highlight the suitability of CARs and TAs for several rounds of reduction/amination reactions in one-pot cascade systems and their potential for the biobased synthesis of terminal amines.

Site-directed mutagenesis and stability of the carboxylic acid reductase MAB4714 from Mycobacterium abscessus.

Carboxylic acid reductases (CARs) catalyze ATP- and NADPH-dependent reduction of carboxylic acids to corresponding aldehydes. Although successful applications of these enzymes for the bioconversion of monocarboxylic acids have already been reported, their applicability for the reduction of dicarboxylic acids is not well understood. Here, we explored the possibility of engineering CARs for enhanced activity toward succinic acid for potential applications in 1,4-butanediol production. Structural models of the carboxylate-binding pocket of the CAR enzyme MAB4714 from Mycobacterium abscessus suggested that its reactivity toward succinic acid could be enhanced by reducing the pocket volume. Using site-directed mutagenesis, we introduced larger side chains into the MAB4714 carboxylate binding pocket and compared the activity of 16 mutant proteins against cinnamic and succinic acids. These experiments revealed that, although the reaction rates remain low, the replacement of Leu284 or Thr285 with Trp increased activity toward succinic acid more than two times. The T285E mutant protein also showed increased activity toward succinic acid, but it was lower than that of T285W. The mutated residues of MAB4714 are located on the flexible loop covering the carboxylate-binding pocket, which appears to contribute to substrate preference of CARs. Thus, reductase activity of CARs against succinic acid can be improved by introducing large side chains into the carboxylate-binding pocket. We also discovered that alanine replacement of the conserved Ser713 in the CAR phosphopantetheine attachment site resulted in complete degradation of the full-length protein into separate A and R domains, suggesting that CAR phosphopantetheinylation is important for its stability in solution.