5 years ago

Computational study of the binding mechanism of medium chain acyl-CoA synthetase with substrate in Methanosarcina acetivorans

The acyl-AMP forming family of adenylating enzymes catalyzes the formation of acyl-CoA from an acyl substrate, ATP, and CoA, which is a metabolite of many catabolic and anabolic processes. The medium-chain acyl-CoA synthetase from Methanosarcina acetivorans, designated MacsMa, uses 2-methylbutyrate as its preferred substrate. It is reported that the interaction between the sidechain of Cys298 and Lys256 of this enzyme is important for the catalytic activity. The mutation of these residues resulted in the changes of the structure stability and the reduced or absence catalytic activity. In the present study, the binding mechanism between the substrate 2-methylbutyrate- AMP (2MeBA) and MacsMa were explored by integrating multiple computational methods including molecular docking, molecular dynamics simulations, binding free energy calculation, active site access channel analysis and principal component analysis. The binding free energy between WT, mutated Macs and substrate was calculated by MM-GBSA method, which indicated that the binding affinity between this enzyme and substrate was stronger in the WT than that in the mutated forms (K256L, K256T and C298Y). Per-residue binding free energy decomposition identified some residues, such as Gly327, Phe350, Gly351, Gln352 and Lys461, which are important for the enzyme and substrate binding affinity. The access channels of the mutant system (MacsK256L, MacsK256T and MacsC298Y) were found to be different from those in the wild-type systems. It suggested that K256L and C298Y induced larger flexibility to the overall protein compared with the WT, whereas K256T induced larger flexibility to the partial protein compared with the WT by PCA vector porcupines. This study provides novel insight to understand the substrate binding mechanism of Macs and useful information for the rational enzyme design.

Publisher URL: www.sciencedirect.com/science

DOI: S016816561731550X

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