Crystal Structural Analysis Of Acetyl Esterase BT4096 From Bacteroides Thetaiotaomicron

Diltiazem is a non-dihydropyridine calcium blocker,which has the characteristics of high selectivity,high tolerance and wide applicability,and has been widely used in the treatment of cardiovascular diseases such as hypertension,angina pectoris and arrhythmia.The drug is metabolized in vivo into multiple metabolites that maintain a variable inhibition of calcium channels.The deacetylation of both diltiazem and diltiazem metabolites in the gut depends on BT4096（GenBank,NP₈13007.1）derived from intestinal bacteria Bacteroides thetaiotaomicron.In this study,BT4096 was expressed and purified,and its crystal structure was solved.BT4096 without the signal peptide contains 440 amino acid residues,which molecular weight is about 51 kDa.The gene encoding BT4096 was synthesized and cloned into the pET 32a vector,and then transferred into the engineering strain BL21（DE3）for expression.The combined protein was purified with two-step Ni-NTA affinity chromatography.Initial crystallization screening was performed by using more than one thousand different compositions in a way of sitting-drop vapor diffusion.The X-ray diffraction datasets were collected at National Synchrotron Radiation Research Center.The phase was solved by soaking Hg-derivatives（ethylmercury chloride）of the crystal.The structure of BT4096 was solved（2.33 A）by single wavelength anomalous dispersion with PHENIX software.Manual adjustments of the models were carried out by using Coot.The protein folds into two parts.The N-terminal part contains the residues Ser18,Asp 194 and His 197 characteristic of a SGNH hydrolase.The hydroxyl O atom in S18 acts as nucleophile to attack the carbonyl C atom of the substrate diltiazem,following the hydrolysis reaction.The C-terminal part is made up of a β-barrel subdomain and an α-helical subdomain,which could enhance substrate binding.Structural comparison shows that the β-barrel subdomain is similar in topology to the immunoglobulin-like domains.This study successfully reveals the structure of a novel acetyl esterase from gut bacteria and shares a plausible binding mode of diltiazem,which is of great biological significance to lay a foundation for the research of other SGNH hydrolases and provide a theoretical gut microbial metabolism model for diltiazem.