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Insights into controlling role of substitution mutation, E315G on thermostability of a lipase cloned from metagenome of hot spring soil.

ABSTRACT: Rational mutagenesis was performed (at the vicinity of the active site residues D317 and H358 of a mature polypeptide) to investigate the role of amino acids in the thermostability/activity of a lipase enzyme. The single variant enzyme created with E315G (lip M2) mutation near one of the active site residue (D317) found to be an important residue in controlling the thermal stability, the variant with E315G mutation demonstrated biochemical properties similar to that of native lipase. However, we found that this mutation strongly affected the activity and stability of the lip M1 mutant, reported in our previous study (Sharma et al. in Gene 491:264-271, 2012b). The dual mutant with E315G/N355K mutation in the Wt showed small increase in the protein thermostability compared to the native lipase, however, the thermostability of the mutant lip M1 was reduced several fold. Presumably, E315G (lip M2) mutation reverted the thermostability evolved by N355K (lip M1). The native and variant enzymes also displayed large variation in enzyme kinetics and their preference for pNP-esters (substrates). We further generated 3D models and studied the loop modelling of the WT and variants. Interestingly, loop region Leu314-Asn321 showed structural flexibility on introducing E315G mutation in the native lipase. On the other hand, lysine in mutant N355K exhibited side chain conformational changes in the loop Thr353-His358 which resulted in its H-bonding with Glu284. In addition, replacing glutamic acid by glycine at 315 position in lip M3 distorted the electrostatic interactions between Glu315 and Lys355 in the flexible loop region Leu314-Asn321.

SUBMITTER: Sharma PK

PROVIDER: S-EPMC3964248 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study describes the phylogenomic analysis and metabolic insights of metagenome-assembled genomes (MAGs) retrieved from hot spring sediment samples. The metagenome-assembled sequences recovered three near-complete genomes belonging to the archaeal phylum. Analysis of genome-wide core genes and 16S rRNA-based phylogeny placed the ILS200 and ILS300 genomes within the uncultivated and largely understudied bathyarchaeal phylum, whereas ILS100 represented the phylum Thaumarchaeota. The average nucleotide identity (ANI) of the bin ILS100 was 76% with Nitrososphaeria_archaeon_isolate_SpSt-1069. However, the bins ILS200 and ILS300 showed ANI values of 75% and 70% with Candidatus_Bathyarchaeota_archaeon_isolate_DRTY-6_2_bin_115 and Candidatus_Bathyarchaeota_archaeon_BA1_ba1_01, respectively. The genomic potential of Bathyarchaeota bins ILS200 and ILS300 showed genes necessary for the Wood-Ljungdahl pathway, and the gene encoding the methyl coenzyme M reductase (mcr) complex essential for methanogenesis was absent. The metabolic potential of the assembled genomes included genes involved in nitrogen assimilation, including nitrogenase and the genes necessary for the urea cycle. The presence of these genes suggested the metabolic potential of Bathyarchaeota to fix nitrogen under extreme environments. In addition, the ILS200 and ILS300 genomes carried genes involved in the tricarboxylic acid (TCA) cycle, glycolysis, and degradation of organic carbons. Finally, we conclude that the reconstructed Bathyarchaeota bins are autotrophic acetogens and organo-heterotrophs. IMPORTANCE We describe the Bathyarchaeota bins that are likely to be acetogens with a wide range of metabolic potential. These bins did not exhibit methanogenic machinery, suggesting methane production may not occur by all subgroup lineages of Bathyarchaeota. Phylogenetic analysis support that both ILS200 and ILS300 belonged to the Bathyarchaeota. The discovery of new bathyarchaeotal MAGs provides additional knowledge for understanding global carbon and nitrogen metabolism under extreme conditions.

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Insights into controlling role of substitution mutation, E315G on thermostability of a lipase cloned from metagenome of hot spring soil.

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