Structure of the glycosyltransferase EryCIII in complex with its activating P450 homologue EryCII

Abstract

In the biosynthesis of the clinically important antibiotic erythromycin D, the glycosyltransferase (GT) EryCIII, in concert with its partner EryCII, attaches a nucleotide-activated sugar to the macrolide scaffold with high specificity. To understand the role of EryCII, we have determined the crystal structure of the EryCIII·EryCII complex at 3.1 Å resolution. The structure reveals a heterotetramer with a distinctive, elongated quaternary organization. The EryCIII subunits form an extensive self-complementary dimer interface at the center of the complex, and the EryCII subunits lie on the periphery. EryCII binds in the vicinity of the putative macrolide binding site of EryCIII but does not make direct interactions with this site. Our biophysical and enzymatic data support a model in which EryCII stabilizes EryCIII and also functions as an allosteric activator of the GT.

Graphical abstract

Fig. 1

Biosynthesis of erythromycin D (3) from the substrates MycEB (1) and TDP-d-desosamine (2) by the GT-auxiliary protein pair EryCIII·EryCII.

Fig. 2

EryCII is necessary for EryCIII activity. Electrospray ionization-LC-MS analysis of the activity of co-expressed EryCIII·EryCII in cell-free extracts. Total ion count (TIC) of an ethyl acetate extract of the GT activity assay (a). Ion trace (m/z = 569) of [M + Na⁺]⁺ of MycEB (b) and (c) ion trace (m/z = 704) of [M + H]⁺ of the product erythromycin D. (d) Plot of peak areas of the erythromycin D product for the control (pET28) and extracts in which only EryCII or EryCIII were expressed are shown. The activity of a mixture of individually expressed EryCIII and EryCII is almost the same as that obtained by co-expression (∗).

Fig. 3

EryCII stabilizes the oligomeric state of EryCIII. Sedimentation velocity profiles, the residuals after fitting and the c(S) distribution for isolated EryCIII (a) and the complex formed between EryCII and EryCIII (b). The r.m.s.d. of the fits and the recovered frictional coefficients were 0.005, 1.7 (a) and 0.03, 1.5 (b), respectively. The sedimentation coefficient distribution of EryCIII reveals higher-order oligomers in addition to the monomeric species (M), which has a sedimentation coefficient of 2.44 S, while that of the EryCIII·EryCII complex consists of a single dominant component at 7.58 S corresponding to a molecular mass of 188 kDa (See also Fig. S3).

Fig. 4

Assembly of the EryCIII·EryCII complex. The biological unit is composed of two EryCIII·EryCII dimers arranged in an almost linear array (a). The EryCIII·EryCII interface is predominantly electrostatic (b and c), while the homodimeric EryCIII·EryCIII interface is hydrophobic (d).

Fig. 5

Topology and structural features of EryCIII and EryCII. (a) Superposition of EryCIII and UrdGT2 (PDB code 2P6P) reveals common features of the GT-B family of GTs. Regions containing the Rossmann-like domains are shown within rectangles. (b) Residues on EryCIII predicted to be involved in donor (d) and acceptor (a) binding are depicted in red and blue, respectively. The A″ and B helices of EryCII interact with helices that are close to the acceptor site of EryCIII. (c) Superposition of EryCII and the cytochrome P450 homologue CYP125 (PDB code 2XC3) showing the A″ helix of EryCII. Although EryCII lacks the heme moiety, the long I helix and others (J, K, K′ and L) in the vicinity of the heme-binding pocket are conserved. (d) Distribution of crystallographic B-factors for the EryCIII·EryCII complex. High B-factor values are observed for residues distal to the A″ helix of EryCII (See also Fig. S5).

Fig. 6

Model of a possible mechanism by which EryCII activates EryCIII. In the absence of EryCII, the dynamics of the N-terminal (green) and C-terminal (grey) domains of EryCIII are such that the acceptor (MycEB) and donor (TDP-desosamine) sites are predominantly disengaged. EryCII binding engages these sites and allows efficient glycosyltransfer. The low activity of some isolated GTs suggests that substrate binding to the enzyme may not be totally dependent on the presence of its auxiliary partner.