Cell-free reduction of carboxylic acids with secreted carboxylic acid reductase.

Mycobacterium marinum CAR (MmCAR) is one of the most widely used CARs as the key enzyme for the synthesis of aldehydes, alcohols and further products from the respective carboxylic acids. Herein, we describe the first functionally secreted 131 kDa CAR and its isolated A-domain using Komagataella phaffii and a methanol-free constitutive expression strategy. Precipitated and lyophilized MmCAR (500 µg) was isolated from the culture supernatant and showed no decrease in activity for piperonylic acid (80% conversion), even when stored for up to 3 weeks at 4°C. Lyophilized MmCAR precipitate gave 48% yield of E/Z-nonanal-4-nitrobenzoyloxime from the reduction of nonanoic acid and in-situ derivatization with O-4-nitrobenzoyl-hydroxylamine. Furthermore, K. phaffii could successfully secrete the MmCAR adenylation domain. Its activity was confirmed by the amidation of benzoic acid with n-hexylamine. Neither enzyme variant was glycosylated by the yeast. In summary, functional CAR can be secreted by K. phaffii and used for cell free conversion of carboxylic acids to various products.

Understanding the riddle of amine oxidase flavoenzyme reactivity on the stereoisomers of N-methyl-dopa and N-methyl-tyrosine.

Enzymes are usually stereospecific against chiral substrates, which is commonly accepted for the amine oxidase family of enzymes as well. However, the FsqB (fumisoquin biosynthesis gene B) enzyme that belongs to the family of sarcosine oxidase and oxidizes L-N-methyl-amino acids, shows surprising activity for both enantiomers of N-methyl-dopa. The aim of this study is to understand the mechanism behind this behavior. Primary docking experiments showed that tyrosine and aspartate residues (121 and 315 respectively) are located on the ceiling of the active site of FsqB and may play a role in fixing the N-methyl-dopa via its catechol moiety and allowing both stereoisomers of this substrate to be in close proximity of the N5 atom of the isoalloxazine ring of the cofactor. Three experimental approaches were used to prove this hypothesis which are: (1) studying the oxidative ability of the variants Y121F and D315A on N-methyl-dopa substrates in comparison with N-methyl-tyrosine substrates; (2) studying the FsqB WT and variants catalyzed biotransformation via high-performance liquid chromatography (HPLC); (3) molecular dynamics simulations to characterize the underlying mechanisms of the molecular recognition. First, we found that the chemical characteristics of the catechol moiety of N-methyl-dopa are important to explain the differences between N-methyl-dopa and N-methyl-tyrosine. Furthermore, we found that Y121 and D315 are specific in FsqB and not found in the model enzyme sarcosine oxidase. The on-bench and theoretical mutagenesis studies show that Y121 residue has a major role in fixing the N-methyl-dopa substrates close to the N5 atom of the isoalloxazine ring of the cofactor. Simultaneously, D315 has a supportive role in this mechanism. Jointly, the experimental and theoretical approaches help to solve the riddle of FsqB amine oxidase substrate specificity.

The family of sarcosine oxidases: Same reaction, different products.

The subfamily of sarcosine oxidase is a set of enzymes within the larger family of amine oxidases. It is ubiquitously distributed among different kingdoms of life. The member enzymes catalyze the oxidization of an N-methyl amine bond of amino acids to yield unstable imine species that undergo subsequent spontaneous non-enzymatic reactions, forming an array of different products. These products range from demethylated simple species to complex alkaloids. The enzymes belonging to the sarcosine oxidase family, namely, monomeric and heterotetrameric sarcosine oxidase, l-pipecolate oxidase, N-methyltryptophan oxidase, NikD, l-proline dehydrogenase, FsqB, fructosamine oxidase and saccharopine oxidase have unique features differentiating them from other amine oxidases. This review highlights the key attributes of the sarcosine oxidase family enzymes, in terms of their substrate binding motif, type of oxidation reaction mediated and FAD regeneration, to define the boundaries of this group and demarcate these enzymes from other amine oxidase families.