Integrative metagenomic and biochemical studies on rifamycin ADP-ribosyltransferases discovered in the sediment microbiome

Abstract

Antibiotic resistance is a serious and growing threat to human health. The environmental microbiome is a rich reservoir of resistomes, offering opportunities to discover new antibiotic resistance genes. Here we demonstrate an integrative approach of utilizing gene sequence and protein structural information to characterize unidentified genes that are responsible for the resistance to the action of rifamycin antibiotic rifampin, a first-line antimicrobial agent to treat tuberculosis. Biochemical characterization of four environmental metagenomic proteins indicates that they are adenosine diphosphate (ADP)-ribosyltransferases and effective in the development of resistance to FDA-approved rifamycins. Our analysis suggests that even a single residue with low sequence conservation plays an important role in regulating the degrees of antibiotic resistance. In addition to advancing our understanding of antibiotic resistomes, this work demonstrates the importance of an integrative approach to discover new metagenomic genes and decipher their biochemical functions.

Figure 1

Integrated analysis of rifamycin ADP-ribosyltransferase (Arr). (a) The sequence conservation of rifamycin ADP-ribosyltransferase (Arr). The residues that are conserved more than 78% of the sequences are highlighted based on the clustalw color scheme. The genes selected for experimental validation are shown as wd1-wd4. (b) Proposed key residues and their molecular interactions in the rifampin-binding sites of Arr-ms structure (PDB 2HW2). In Arr-ms, the variable X residue in M[R|K][D|E]XL motif is Gly129, and its position is highlighted with a red arrow. Water molecules are depicted with spheres.

Figure 2

Structural analysis of rifamycin ADP-ribosyltransferase (Arr). (a) A structure of rifampin. The aliphatic ansa chain and the site of modification by ADP-ribosyltransferase is colored in blue and red, respectively. (b) Two substrate-binding pockets depicted in the structure of rifamycin ADP-ribosyltransferase from arr-ms (PDB 2HW2). The variable X residue in M[R|K][D|E]XL motif is  highlighted with a red arrow.

Figure 3

A phylogenetic tree of the metagenomic arr genes and well-known arr genes. Red circles denote arr genes collected from the metagenomic sample in this work. Red filled circles denote arr genes validated experimentally in this work. Blue rectangles denote previously known and experimentally validated arr genes. Green triangles denote arr genes referenced previously but not validated experimentally.

Figure 4

Biochemical analysis of Arr-wd 1–4 proteins. (a) A representative HPLC trace of rifampin ADP-ribosylation by Arr-wd proteins. (b–d) Steady-state ADP-ribosyltransferase activities of Arr-wd proteins together with the reported values of Arr proteins. (b) Turnover numbers, k_cat (s⁻¹) (c) Catalytic efficiencies, k_cat/K_M (s⁻¹ M⁻¹) (d) Michaelis constants, K_M (mM) (e) Dissociation constants, K_D (mM) (f) MIC values (μg/mL). Values reported in (b–d) and (e) are derived from the fit to the Michaelis-Menten equation and Stern-Volmer equation, respectively.

Figure 5

Kinetic and thermodynamic parameters of Arr-wd proteins from the steady-state reactions with rifampin derivatives, rifampin, rifapentine, rifaximin, and rifabutin. (a) Turnover numbers, k_cat (s⁻¹) (b) Catalytic efficiencies, k_cat/K_M (s⁻¹ M⁻¹) (c) Michaelis constants, K_M (mM) (d) Dissociation constants, K_D (μM). Values reported in (a–c) and (d) are derived from the fit to the Michaelis-Menten equation and Stern-Volmer equation, respectively.

Figure 6

Kinetic and thermodynamic parameters of Arr-wd3 H125 variants from the steady-state reactions with rifampin derivatives. (a) Turnover numbers, k_cat (s⁻¹) (b) Catalytic efficiencies, k_cat/K_M (s⁻¹ M⁻¹) (c) Michaelis constants, K_M (mM) (d) Dissociation constants, K_D (μM). Values reported in (a–c) and (d) are derived from the fit to the Michaelis-Menten equation and Stern-Volmer equation, respectively.