Structural Characterization And Bioinformatic Analysis Of Acetyl-CoA Carboxylase (AccB) From Streptomyces Antibioticus

Acetyl-CoA carboxylase(ACC)is crucial for polyketides biosynthesis and acts as an essential metabolic checkpoint.It is also an attractive drug target against obesity,cancer,microbial infections,and diabetes.However,the lack of knowledge,particularly sequence-structure function relationship to narrate ligand-enzyme binding,has hindered the progress of ACC-specific therapeutics and unnatural “natural” polyketides.Structural characterization of such enzymes will boost the opportunity to understand the substrate binding,designing new inhibitors and information regarding the molecular rules which control the substrate specificity of ACCs.To understand the substrate specificity,we determined the crystal structure of Acc B(Carboxyl-transferase,CT)from Streptomyces antibioticus with a resolution of 2.3 (?) and molecular modeling approaches were employed to unveil the molecular mechanism of acetyl-CoA recognition and processing.The CT of S.antibioticus shares a similar structural organization with the previous structures and the two steps reaction was confirmed by enzymatic assay.Furthermore,to reveal the key hotspots required for the substrate recognition and processing,in silico mutagenesis validated only three key residues(V223,Q346,and Q514)that help in the fixation of the substrate.Moreover,we also presented atomic level knowledge on the mechanism of the substrate binding,which unveiled the terminal loop(500-514)function as an opening and closing switch and pushes the substrate inside the cavity for stable binding.A significant decline in the hydrogen bonding half-life was observed upon the alanine substitution.Consequently,the presented structural data highlighted the potential key interacting residues for substrate recognition and will also help to re-design ACCs active site for proficient substrate specificity to produce diverse polyketides.Malonyl-CoA serves as the main building block for the biosynthesis of many important polyketides,as well as fatty acids derived compounds,such as biofuel.Escherichia coli,Corynebacterium gultamicum,and Saccharomyces cerevisiae have recently been engineered towards the biosynthesis of such compounds.However,the developed processes and strains often have insufficient productivity.In the current study we used in silico enzymes engineering approach to improve the binding of acetyl-CoA with Acetyl-CoA carboxylase(ACC).We generated different mutations and the impact was calculated which reported that three mutations i.e.,S343 A,T347W and S350 F significantly improve the substrate binding.Molecular docking investigation revealed altered binding network compared to the wild type.In mutants,additional interactions stabilize the binding of the inner tail of acetyl-CoA.Using molecular simulation,the stability,compactness,hydrogen bonding and proteins motions were estimated,revealing different dynamic properties owned by the mutants only but not by the wild type.The findings were further validated by using binding free energy method,which revealed these mutations as favorable substitutions.The total binding free energy was reported to be-52.66±0.11 kcal/mol for the wild type,-55.87±0.16kcal/mol for the S343 A mutant,-60.52±0.25 kcal/mol for T347 W mutant,and-59.64±0.25kcal/mol for the S350 F mutant.This shows that the binding of the substrate is increased due to the induced mutations and strongly corroborates with the docking results.In sum,this study provides basis for improved substrate binding and potential application in industrial processes.