Role of an Active Site Loop in the Promiscuous Activities of Amycolatopsis sp. T-1-60 NSAR/OSBS

Abstract

The o-succinylbenzoate synthase (OSBS) family is part of the functionally diverse enolase superfamily. Many proteins in one branch of the OSBS family catalyze both OSBS and N-succinylamino acid racemization in the same active site. In some promiscuous NSAR/OSBS enzymes, NSAR activity is biologically significant in addition to or instead of OSBS activity. Identifying important residues for each reaction could provide insight into how proteins evolve new functions. We have made a series of mutations in Amycolatopsis sp. T-1-60 NSAR/OSBS in an active site loop, referred to as the 20s loop. This loop affects substrate specificity in many members of the enolase superfamily but is poorly conserved within the OSBS family. Deletion of this loop decreased OSBS and NSAR catalytic efficiency by 4500-fold and 25,000-fold, respectively, showing that it is essential. Most point mutations had small effects, changing the efficiency of both NSAR and OSBS activities <10-fold compared to that of the wild type. An exception was F19A, which reduced kcat/KM(OSBS) 200-fold and kcat/KM(NSAR) 120-fold. Mutating the surface residue R20E, which can form a salt bridge to help close the 20s loop over the active site, had a more modest effect, decreasing kcat/KM of OSBS and NSAR reactions 32- and 8-fold, respectively. Several mutations increased KM of the NSAR reaction more than that of the OSBS reaction. Thus, both activities require the 20s loop, but differences in how mutations affect OSBS and NSAR activities suggest that some substitutions in this loop made a small contribution to the evolution of NSAR activity, although additional mutations were probably required.

Enzyme Promiscuity in Enolase Superfamily. Theoretical Study of o-Succinylbenzoate Synthase Using QM/MM Methods

“…However, it must be pointed out that mutations on the loop 20 of Amycolatopsis OSBS/NAAAR enzyme reduced both the synthase and the racemase activities, suggesting that evolution of this loop were not the only changes needed to evolve the latter activity. 57 As discussed below other residues contribute to accelerate the racemase activity in Amycolatopsis OSBS/NAAAR. We can analyze the contribution of the interaction energy of each residue to the barrier (calculated using Eq.…”

N-succinylamino acid racemases: Enzymatic properties and biotechnological applications

“…After deciphering the natural function of NSAR, huge efforts have been paid on finding the molecular determinants allowing the evolution of the OSBS family and to understand the substrate promiscuity of NSAR/OSBS enzymes [69][70][71][72][73][74]. Protein…”

“…works have proved the difficulties to find the determinant allowing the evolution of OSBS family towards NSAR activity [70,71,74]. NSAR activity thus appears to be a recent evolutionary event towards new metabolic activities such as the interconversion of D-to L-amino acids for Geobacillus kaustophilus, which to date seems to be confined principally to enzymes in the Firmicutes NSAR/OSBS subfamily [57,58,74].…”

“…NSAR was already shown to be a member of the functional diverse enolase superfamily before the beginning of the new millennium [51]. Several works at the beginning of the 21 st century focused on deciphering the natural function of NSARs and some evolutionary aspects [51,57,58,[69][70][71][72][73][74][75][76] (see below), allowing to localize new NSARs in Bacillus/Geobacillus genera. Some of the NSARs belonging to this genera have been highly characterized and/or applied to the production of optically pure amino acids [27][28][29][30][31][32][33][34][35][36][37][38]59,[61][62][63][64][65][66][67][68].…”

Inactivation of Mandelate Racemase by 3-Hydroxypyruvate Reveals a Potential Mechanistic Link between Enzyme Superfamilies

“…[6][7][8] Studies of members of the enolase superfamily have afforded important insights into enzyme evolution and developing strategies for assigning the correct functions to proteins identified in genome projects. 3,[9][10][11][12] The well-characterized NOT THE PUBLISHED VERSION; this is the author's final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page.…”