Probing the influence of non-covalent contact networks identified by charge density analysis on the oxidoreductase BacC

Abstract

Bacillus subtilis BacC is an oxidoreductase involved in the biosynthesis of the potent antibiotic bacilysin. The crystal structure of BacC was determined at 1.19 Å resolution. An experimental charge density approach was used to calculate non-covalent interactions within the monomer and across the dimeric interface of BacC. This interaction network, in turn, enabled an analysis of non-covalently connected paths that span the protein structure. One of the pathways of non-covalent interactions was examined by mutational analysis. Biochemical analysis of BacC mutants with potential disruptions in non-covalent interactions along this path revealed that residues that form nodes in pathways of non-covalent interactions influence catalytic activity more than others in a similar chemical environment. Furthermore, we note that nodes in the non-covalent interaction networks are co-localized with compensatory mutation sites identified by multiple sequence alignment of proteins with low sequence similarity to BacC. Put together, this analysis supports the hypothesis that non-covalent nodes represent conserved structural features that can impact the catalytic activity of an enzyme.

Multi‐faceted Set‐up of a Diverse Ketoreductase Library Enables the Synthesis of Pharmaceutically‐relevant Secondary Alcohols

“…The KRED‐catalyzed asymmetric reduction of the α‐ketoester 4 a to ( R )‐ethyl 2‐hydroxy‐4‐phenylbutanoate ( 4 b ), for example, is literature‐known, [16] since ( R )‐ 4 b is pharmaceutically relevant for synthesizing several ACE‐inhibitors [4k] . KREDs exhibiting this industrially desirable enantiospecificity were also identified in our KRED library, with KRED 23, [17] 30, [18] 31, [19] 47, [20] as well as the novel KREDs from P. tolaasii 40 and 50 being able to catalyze the selective reduction of 4 a . Remarkably, the novel KRED 50 showed the highest activity for the selective reduction of 4 a to ( R )‐ 4 b within the library, expanding the biocatalytic toolbox of known KREDs for the synthesis of this chiral reaction intermediate.…”

The Realm of Unconventional Noncovalent Interactions in Proteins: Their Significance in Structure and Function

“…We believe that the revolutionary improvement in the resolution of 3D structural data of proteins determined using cryoEM will aid in the identification of unconventional noncovalent interactions in proteins and reveal new roles of these interactions in protein function. With many more protein structures being determined at very- and ultra-high resolution, use of charge-density analysis combined with detailed quantum chemical calculations, such as Atoms In Molecule analysis, will provide unprecedented insights into the nature and function of noncovalent interactions in proteins. ,, We believe that with newer and improved experimental and computational techniques being used for probing noncovalent interactions in small molecules and proteins, our understanding of the nature of these interactions, their role, and significance is only on the rise.…”

“…With many more protein structures being determined at very- and ultra-high resolution, use of charge-density analysis combined with detailed quantum chemical calculations, such as Atoms In Molecule analysis, will provide unprecedented insights into the nature and function of noncovalent interactions in proteins. 75 , 76 , 182 We believe that with newer and improved experimental and computational techniques being used for probing noncovalent interactions in small molecules and proteins, our understanding of the nature of these interactions, their role, and significance is only on the rise.…”

“…A charge-density analysis of an iron–sulfur protein using X-ray data at an ultrahigh resolution of 0.48 Å revealed the distorted valency density of S atoms of the iron–sulfur cluster and the coordinating cysteine interacting with H of main-chain amides and also those bonded to C of hydrophobic residues of the protein . The analysis indicated modulation in the negative charge of S due to charge transfer from S to the interacting H. Further highlighting the importance of S-mediated H-bonds, charge-density analysis of very-high-resolution X-ray data of the enzyme BacC revealed a network of noncovalent interactions mediated by the thiol group of the three cysteines, which when disrupted by mutagenesis had a significant effect on the enzyme activity . Compensatory mutation re-established the network.…”

“… 75 The analysis indicated modulation in the negative charge of S due to charge transfer from S to the interacting H. Further highlighting the importance of S-mediated H-bonds, charge-density analysis of very-high-resolution X-ray data of the enzyme BacC revealed a network of noncovalent interactions mediated by the thiol group of the three cysteines, which when disrupted by mutagenesis had a significant effect on the enzyme activity. 76 Compensatory mutation re-established the network.…”