New N-Acetyltransferase Fold in the Structure and Mechanism of the Phosphonate Biosynthetic Enzyme FrbF

Abstract

The enzyme FrbF from Streptomyces rubellomurinus has attracted significant attention due to its role in the biosynthesis of the antimalarial phosphonate FR-900098. The enzyme catalyzes acetyl transfer onto the hydroxamate of the FR-900098 precursors cytidine 5-monophosphate-3-aminopropylphosphonate and cytidine 5-monophosphate-N-hydroxy-3-aminopropylphosphonate. Despite the established function as a bona fide N-acetyltransferase, FrbF shows no sequence similarity to any member of the GCN5-like N-acetyltransferase (GNAT) superfamily. Here, we present the 2.0 Å resolution crystal structure of FrbF in complex with acetyl-CoA, which demonstrates a unique architecture that is distinct from those of canonical GNAT-like acetyltransferases. We also utilized the co-crystal structure to guide structure-function studies that identified the roles of putative active site residues in the acetyltransferase mechanism. The combined biochemical and structural analyses of FrbF provide insights into this previously uncharacterized family of N-acetyltransferases and also provide a molecular framework toward the production of novel N-acyl derivatives of FR-900098.

A low-barrier hydrogen bond mediates antibiotic resistance in a noncanonical catalytic triad

“…A water molecule also hydrogen-bonds to the acetyl carbonyl and forms a hydrogen-bonding network with two residues, His 189 and Glu 192 , which have been proposed in other studies to be important in the catalytic mechanism ( Fig. 1 ) ( 8 , 9 ). This water molecule is in the same location as the N3 amine of the sisomicin ( Fig.…”

“…Crystallographic data are summarized in Table 1 . The fold of AAC-VIa places it in a distinct, noncanonical GNAT subfamily, with the overall structure being essentially identical to the other four members whose structures are known to date: YokD (PDB code 2NYG), FrbF [PDB code 3SMA ( 9 )], HMB0038 (PDB code 5HT0), and BA2930 [PDB code 3E4F ( 8 )] (fig. S2).…”

“…The crystal structures of three additional members of this subfamily have also been reported, with all having a similar fold and conservation of proposed catalytically important residues. Yet significant knowledge about the catalytic mechanism is lacking for this pathologically important GNAT subfamily ( 9 ).…”

Characterization and application of the Fe(ii) and α-ketoglutarate dependent hydroxylase FrbJ

“…The recombinant enzyme was codon-optimized for heterologous expression in E. coli and was designed to lack the N-terminal signal peptide. 21–22 Enzymatic activity was monitored by NADPH oxidation at 340 nm, and subsequent inhibition values for the IC 50 and K I were obtained for each of the prospective antimalarial compounds. Similar to the in vivo bioassays, only the free, unmethylated phosphonate compounds demonstrated in vitro inhibition of pfDxr.…”

Phosphonate biosynthesis and catabolism: a treasure trove of unusual enzymology

“…The late steps of FR-900098 biosynthesis have also been characterized in vitro [33,34]. Perhaps the most interesting enzyme is the bifunctional enzyme FrbH.…”