| A variety of acyl substitution reactions such as fatty acyl and aromatic acyl occur frequently in living organisms,and the BAHD superfamily,the most representative family of acyltransferases in plants,plays a rather important role in plants.The p HBMT1 belonging to the BAHD superfamily is responsible for catalyzing the phydroxybenzoylation of monolignans.Para-hydroxybenzoic acid has a wide range of applications in a variety of ways,including as a raw material for chemical synthesis,a food additive,and a pesticide intermediate.In order to deeply understand its function,we attempted to investigate its three-dimensional structure by means of structural biology to understand its spatial conformational changes before and after binding to substrates,and to provide a theoretical basis for the design of an efficient catalytic industrial enzyme protein.The protease was successfully expressed using the prokaryotic expression system,and the high-purity p HBMT1 enzyme protein was subsequently obtained by nickel column affinity chromatography,ion exchange chromatography and molecular sieve chromatography in turn,and the purity was 98% by SDS-PAGE electrophoresis.After obtaining a large amount of high-purity,highly homogeneous p HBMT1 protein,the protein crystals were initially screened using a crystal screening kit purchased from Hamptons.The crystals were finally screened in pool solution #33 in PEG/Ion,and after optimization,single crystal data were collected using an X-ray crystal diffractometer to confirm a resolution of approximately 2.8 ?.The cell parameters were a=62.9,b=83.8,and c=173.2,respectively,with crystals in a trigonal lattice.It was predicted that 41 L,165H,169 D,and 369 K might be its active centers,and mutations were performed for the corresponding sites. |
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