Roles of the second-shell amino acid R266 in other members of the MLE subgroup of the enolase superfamily
biorxiv(2022)
摘要
Catalytic promiscuity is the coincidental ability to catalyze non-biological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies such a pre-adaptive residue in an N -succinylamino acid racemase/ o -succinylbenzoate synthase (NSAR/OSBS) enzymes from the NSAR/OSBS subfamily. Previously, we identified a point mutation, R266Q, in the catalytically promiscuous Amycolatopsis sp. T-1-60 NSAR/OSBS that has a deleterious effect on NSAR activity with a lesser effect on OSBS activity (Truong et al ., in preparation). We demonstrated that R266 was a pre-adaptive feature that enabled the emergence and evolution of NSAR activity in AmyNSAR/OSBS. We examined the role of the residue R266 in the evolution of NSAR activity by examining the effects of the single substitution R266Q in other members of the NSAR/OSBS subfamily including Enterococcus faecalis NSAR/OSBS, Roseiflexus castenholzii NSAR/OSBS, Lysinibacillus varians NSAR/OSBS, and Listeria innocua NSAR/OSBS, which have been previously characterized to carry out both OSBS and NSAR activities efficiently. RcNSAR/OSBS, LvNSAR/OSBS, EfNSAR/OSBS, and LiNSAR/OSBS are 49, 48, 32, and 28% identical, respectively, to AmyNSAR/OSBS. We found that while the R266Q mutation decreases NSAR activity more than OSBS activity, as expected, in most NSAR/OSBS members, the differential effects of the R266Q substitution on NSAR and OSBS activities are not as striking as observed in AmyNSAR/OSBS. In some homologs, the R266Q mutation has very deleterious effects on both OSBS and NSAR activities. Furthermore, the mutation unexpectedly decreases OSBS activity more than NSAR activity in LiNSAR/OSBS. Thus, the effects of R266Q on NSAR and OSBS activities depend on differences in sequence context between members of the NSAR/OSBS subfamily, demonstrating the complex role of epistasis in the evolution of NSAR activity in the NSAR/OSBS subfamily.
### Competing Interest Statement
The authors have declared no competing interest.
* OSBS
: o -succinylbenzoate synthase
NSAR
: N -succinylamino acid racemase
EfNSAR/OSBS
: Enterococcus faecalis NSAR/OSBS
RcNSAR/OSBS
: Roseiflexus castenholzii NSAR/OSBS
LvNSAR/OSBS
: Lysinibacillus varians NSAR/OSBS
LiNSAR/OSBS
: Listeria innocua NSAR/OSBS
kex
: exchange rate
DE
: Dipeptide Epimerase
NSPG
: N -succinylphenylglycine
SHCHC
: 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate
DSF
: Differential Scanning Fluorimetry
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关键词
enolase,mle subgroup,second-shell
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