Relationships between Substrate Promiscuity and Chiral Selectivity of Esterases from Phylogenetically and Environmentally Diverse Microorganisms

Abstract

Substrate specificity and selectivity of a biocatalyst are determined by the protein sequence and structure of its active site. Finding versatile biocatalysts acting against multiple substrates while at the same time being chiral selective is of interest for the pharmaceutical and chemical industry. However, the relationships between these two properties in natural microbial enzymes remain underexplored. Here, we performed an experimental analysis of substrate promiscuity and chiral selectivity in a set of 145 purified esterases from phylogenetically and environmentally diverse microorganisms, which were assayed against 96 diverse esters, 20 of which were enantiomers. Our results revealed a negative correlation between substrate promiscuity and chiral selectivity in the evaluated enzymes. Esterases displaying prominent substrate promiscuity and large catalytic environments are characterized by low chiral selectivity, a feature that has limited commercial value. Although a low level of substrate promiscuity does not guarantee high chiral selectivity, the probability that esterases with smaller active sites possess chiral selectivity factors of interest for industry (>25) is significantly higher than for promiscuous enzymes. Together, the present study unambiguously demonstrates that promiscuous and selective esterases appear to be rare in nature and that substrate promiscuity can be used as an indicator of the chiral selectivity level of esterases, and vice versa.

Ancestral Sequence Reconstruction Enhances Gene Mining Efforts for Industrial Ene Reductases by Expanding Enzyme Panels with Thermostable Catalysts

“…Individual enzyme enantiomeric excess data evaluated by GC chromatography is included in our master data Tables S2.1 and S2.2. A previous study that included a panel of 145 wild-type microbial esterases unambiguously concluded that there is a direct relationship between enzyme enantioselectivity and substrate specificity . In addition, ancestral enzymes are generally considered to have a broader substrate scope compared to enzymes sourced from extant genes .…”

“…A previous study that included a panel of 145 wild-type microbial esterases unambiguously concluded that there is a direct relationship between enzyme enantioselectivity and substrate specificity. 35 In addition, ancestral enzymes are generally considered to have a broader substrate scope compared to enzymes sourced from extant genes. 36 The enantioselectivity results of our 101enzyme panel when tested with substrate 1a support this general scientific consensus.…”

Biocatalysis and Biotransformations

“…Finally, in the present issue, Coscolín and coworkers [36] and Nguyen and coworkers [37] investigated the properties and catalytic performance of biocatalysts newly isolated from genomes and metagenomes. In particular, Coscolín and coworkers investigated an ample set of metagenomicsderived esterases and lipases to reveal the extent by which promiscuity and selectivity co-exist in these catalytically-important biocatalysts, so as to identify chiral selective and promiscuous esterases and lipases.…”

Emerging Technologies for Biocatalysis in the Pharmaceutical Industry

“…A recent report from Pfizer comparing 57 wild-type ERED enzymes and 56 ASR-derived EREDs demonstrated that ASR enzymes were significantly less enantioselective than their wild-type counterparts. 44 Decreased enantioselectivity has been reported to correlate with increased substrate promiscuity, 45 which may indicate that ASR enzymes have broader substrate scopes than wild-type enzymes, but that they are more likely to display lower enantioselectivity.…”