Determination of the structure of selenomethionine-labelled hydroxymethylbilane synthase in its active form by multi-wavelength anomalous dispersion

Abstract

The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2. 4 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.

Anomalous signal indicators in protein crystallography

“…For the amplitude-based criterion, a value of 0.8 or lower indicates no anomalous signal and amplitude-based anomalous signal-to-noise ratios larger than 1.2 indicate the presence of significant signal (Sheldrick, 2004). A quantity related to the average anomalous signal-to-noise ratio is the number or fraction of Bijvoet differences whose absolute value is larger than three times its estimated standard deviation (Hä dener et al, 1999). The latter criterion is very closely related to the measurability as defined by Parthasarathy & Parthasarathi (1974) as discussed in x3.4.…”

Structural Insights into E. coli Porphobilinogen Deaminase during Synthesis and Exit of 1-Hydroxymethylbilane

“…Five crystal structures of E. coli PBGD ( EcPBGD): 1GTK [9], 1YPN [10], 2YPN [11], 1AH5 [12], 1PDA [8]; two of hPBGD: 3ECR [13], 3EQ1 [5]; and most recently, one of Arabidopsis thaliana PBGD (AtPBGD): 4HTG [14] are available in the protein data bank (PDB) [15]. The structure of EcPBGD (Figure 2A) consists of 3 domains of α/β class.…”

Dibenzyl 5-(2-chloroethyl)-3,7-diethyl-2,8-dimethyldipyrromethane-1,9-dicarboxylate

“…Their chemical synthesis in the laboratory often involves the so-called 2 + 2 synthesis, where two dipyrromethane units are condensed (Lindsey, 2000). In nature, porphyrins are prepared by attaching pyrrole units to a dipyrromethane co-factor of porphobilinogen deaminase (Louie et al, 1992;Ha È dener et al, 1999). Dipyrromethane units are structural building blocks of many bilanes (Zhang et al, 1998;Senge et al, 2001) and calixphyrins (Sessler et al, 2003).…”