Biocatalysis, one of the most environmentally friendliest methods for synthesizing chiral synthons, has emerged as a desirable process for manufacturing active pharmaceutical ingredients and agrochemicals, most of which contain one or more chiral amine moieties. Compared with the traditional biocatalytic methods for the synthesis of chiral amines involving lipases or transaminases, enzymatic imine reduction is a more promising approach. The single-step enzymatic reduction of prochiral imines to the corresponding amines can yield 100% of the required enantiomer without any by-products. Furthermore, the reduction of imines generated in situ through the condensation of amines and carbonyl compounds can be used to synthesize almost any primary, secondary, or tertiary amine. In the past decade, several imine-reducing enzyme families, such as Streptomyces imine reductases (IReds), native amine dehydrogenases and engineered leucine/phenylalanine dehydrogenases and opine dehydrogenases, have been explored. The novel ornithine cyclodeaminase/μ-crystallin (OCD/CRYM) superfamily, despite its ubiquitous presence in all three domains of life, has only a few well-studied members. The ornithine cyclodeaminase/µ-crystallin (OCD/CRYM) superfamily consists of proteins capable of imine reduction, which have been relatively unexplored regarding the synthesis of chiral amines.
The current study reports the purification and characterization of a stereospecific imine reductase that belongs to the OCD/CRYM superfamily from the yeast Candida parapsilosis ATCC 7330 (CpIR), a versatile biocatalyst and a rich source of highly stereospecific oxidoreductases. The recombinant gene was overexpressed in Escherichia coli, and the protein CpIR was purified to homogeneity. CpIR catalyzed the alkylamination of α-keto acids/esters producing exclusively (S)-N-alkyl amino acids/esters, e.g. N-methyl-L-alanine with > 90% conversion and > 99% enantiomeric excess (ee). The enzyme showed the highest activity for the alkylamination of pyruvate and methylamine, leading to N-methyl- L-alanine with an apparent KM of 15.04± 2.8 mM and Vmax of 13.75 ± 1.07 μmol/min.mg. CpIR also catalyzed (i) the reduction of imines, e.g. 2-methyl-1-pyrroline to (S)-2-methylpyrrolidine with ∼30% conversion and 75% ee and (ii) the dehydrogenation of cyclic amino acids, e.g. L-Proline (as monitored by reduction of cofactor NADP+ spectrophotometrically). The structure of CpIR was determined at 2.5 A resolution and represents the first structure of a yeast homolog from the OCD/CRYM superfamily. The structure was compared to other OCD/CRYM proteins to help understand structure-function and evolutionary relationships. The overall structure resembles the bacterial homologs of this superfamily, while the active site and biochemical properties are similar to mammalian homologs. Analysis of the kinetic behavior revealed that CpIR followed a sequential tri-substrate kinetic mechanism, with NADPH, Pyruvate, and Methylamine binding in that order. Based on the analysis of the structure and the kinetic behavior, a plausible mechanism for reductive amination is deduced.
Publications:
1. Uma Mahesh, V. N. M., and Chadha, A. (2021). “Imine reduction by an Ornithine cyclodeaminase/μ-crystallin homolog purified from Candida parapsilosis ATCC 7330,” Biotechnology Reports, 31, e00664.
2. Uma Mahesh, V. N. M., Faidh, M. A., and Chadha, A. (2022). “The ornithine cyclodeaminase/µ-crystallin superfamily of proteins: A novel family of oxidoreductases for the biocatalytic synthesis of chiral amines,” Current Research in Biotechnology, 4, 402-419.