Structure of a gut microbial diltiazem-metabolizing enzyme suggests possible substrate binding mode

https://doi.org/10.1016/j.bbrc.2020.04.116Get rights and content

Highlights

  • Structure of diltiazem deacetylase BT4096 from a human gut microbe is solved.

  • The catalytic domain of BT4096 features GDSL/SGNH-family hydrolase.

  • The roles of the unique C-terminal domains are proposed by structural analyses.

Abstract

When administrated orally, the vasodilating drug diltiazem can be metabolized into diacetyl diltiazem in the presence of Bacteroides thetaiotaomicron, a human gut microbe. The removal of acetyl group from the parent drug is carried out by the GDSL/SGNH-family hydrolase BT4096. Here the crystal structure of the enzyme was solved by mercury soaking and single-wavelength anomalous diffraction. The protein folds into two parts. The N-terminal part comprises the catalytic domain which is similar to other GDSL/SGNH hydrolases. The flanking C-terminal part is made up of a β-barrel subdomain and an α-helical subdomain. Structural comparison shows that the catalytic domain is most akin to acetyl-xylooligosaccharide esterase and allows a plausible binding mode of diltiazem to be proposed. The β-barrel subdomain is similar in topology to the immunoglobulin-like domains, including some carbohydrate-binding modules, of various bacterial glycoside hydrolases. Consequently, BT4096 might originally function as an oligosaccharide deacetylase with additional subdomains that could enhance substrate binding, and it acts on diltiazem just by accident.

Introduction

Drug delivery and metabolism in human beings can contribute to the efficacy of medication. Orally administered drugs can be metabolized by intestinal microbiome in addition to the human tissues [1,2]. Several studies demonstrate enzymatic paradigms that gut microbes exploit to influence the in vivo drug metabolism [3,4]. Zimmermann et al. showed that human gut microbes are involved in chemical modifications of orally administered drugs, and the findings could explain interpersonal variation in drug metabolism and toxicity by microbiome difference [4]. In that study Bacteroides thetaiotaomicron was used as an exemplary source species, from which the gene product BT4096 was found to metabolize diltiazem by deacetylation (Fig. 1). Diltiazem is a calcium channel blocker that works by relaxing the smooth muscle of arterial walls. Owing to its vasodilatory effect, diltiazem is used in treating angina pectoris and systemic hypertension. When taken orally, the parent drug diltiazem is converted to its deacetyl metabolite by BT4096 (or other homologues) in human gut [4]. Although both are effective vasodilators, the difference in polarity and solubility may affect the rates of absorption and elimination [5].

The full-length BT4096 is comprised of 460 amino acids, and it is classified as a GDSL/SGNH hydrolase family protein. A BLAST search showed several similar GDSL-like proteins in the Protein Data Bank with 23–25% sequence identity to the N-terminal 220 residues of BT4096. The remaining part, however, did not show significant similarity to any protein with known 3D structure. Despite the varying sizes, all GDSL/SGNH hydrolases contain a catalytic core of about 20 kDa which is made up of a central parallel β-sheet and several flanking α-helices [6]. A GDSL hydrolase features a conserved N-terminal GxSx motif in the catalytic domain that contains the Ser nucleophile, which in the other hydrolases has a rather central location. The SGNH subfamily of GDSL hydrolases has four strictly conserved residues Ser-Gly-Asn-His in four conserved regions I-II-III-V. The first corresponds to the same Ser in the GDSL motif; the second and third constitute the unique oxyanion hole; and the fourth and its preceding Asp in the DxxH motif serve as the other members of the catalytic triad [6].

To elucidate the mechanism of action of BT4096 on the drug diltiazem, we cloned, expressed, purified and crystallized the enzyme in a recombinant form with the first 20 amino acid residues replaced by a His-tag (which was removed in the end). The crystal structure was then solved by single-wavelength anomalous diffraction (SAD) method using a mercury derivative. The protein was found to contain a β-barrel subdomain and an α-helical subdomain as its C-terminal part, in addition to the N-terminal catalytic domain. The structure was compared with those of closely related GDSL/SGNH hydrolases, and possible substrate binding modes were investigated.

Section snippets

Cloning, expression and purification of recombinant BT4096

The gene encoding BT4096 from B. thetaiotaomicron (GenBank WP_008760980.1) without the 20-residue signal peptide but with an N-terminal TEV protease site and a linker (AGAGAGAGA) was chemically synthesized and cloned to the pET-32a vector by Sangon Biotechnology (Shanghai, China). The plasmid was transformed to Escherichia coli BL21(DE3). After screening, a single colony was propagated overnight at 37 °C in lysogeny broth medium (LB) that contained 100 μg/mL ampicillin. Ten liters of fresh LB

Crystal structure of BT4096

By using the SAD data set and the PHENIX software, an initial model of 1528 amino acid residues for the four BT4096 monomers were obtained. By exploiting the four-fold NCS relationship between the monomers, a continuous model for the 440-residue polypeptide chain of BT4096 was constructed, along with part of the N-terminal expression tag. Preliminary refinement of four copies of this model against the SAD data gave R and Rfree values of 0.222 and 0.262. However, some regions appeared to be

Concluding remarks

In this study we determined the crystal structure of BT4096, an acetyl esterase from the human gut microbe B. thetaiotaomicron that converts the drug diltiazem to its deacetyl metabolite. The protein folds into an N-terminal catalytic domain, which is similar to the carbohydrate degrading acetylesterase Axe2, and the flanking C-terminal β-barrel and α-helical subdomains. The latter are supposed to provide additional interactions with substrate. Judging by the presence of a wide groove that

Declaration of competing interest

The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgement

This work has been supported by the National Key Research and Development Program of China (2019YFA070030), the National Natural Science Foundation of China (31870790, 31900930 and 31971205), KFZD-SW-215-01 from CAS, the Natural Science Foundation of Tianjin (18JCYBJC24300) and the Taiwan Protein Project (Grant No. AS-KPQ-109-TPP2). We thank NSRRC (National Synchrotron Radiation Research Center, Taiwan) for access to beam line TPS-05 A that contributed for the synchrotron data collection.

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