The crystal structure of Escherichia coli class II fructose-1,6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity

The structure of a class II fructose-1,6-bisphosphate aldolase in complex w ith the substrate analogue and inhibitor phosphoglycolohydroxamate (PGH) ha s been determined using X-ray diffraction terms to a resolution of 2.0 Angs trom (1 Angstrom = 0.1 nm). The crystals are trigonal, space group P3(1)21 with a = b = 78.24 Angstrom, c = 289.69 Angstrom. The asymmetric unit is a homodimer of (alpha/beta)(8) barrels and the model has refined to give R-wo rk 19.2%, R-free (based on 5% of the data) 23.0%. PGH resembles the ene-dio late transition state of the physiological substrate dihydroxyacetone phosp hate. It is well ordered and bound in a deep polar cavity at the C-terminal end of the (alpha/beta)(8) barrel, where it chelates the catalytic zinc io n using hydroxyl and enolate oxygen atoms. Trigonal bipyramidal coordinatio n of the zinc ion is completed by three histidine residues. The complex net work of hydrogen bonds at the catalytic centre is required to organise the position of key functional groups and metal ion ligands. A well-defined mon ovalent cation-binding site is observed following significant re-organisati on of loop structures. This assists the formation of a phosphate-binding si te on one side of the barrel that tethers PGH in the catalytic site. The po sitions of functional groups of substrate and putative interactions with ke y amino acid residues are identified. Knowledge of the complex structure co mplements the results of spectroscopic and site-directed mutagenesis studie s, and contributes to our understanding of the mechanism and substrate spec ificity of this family of enzymes. A reaction mechanism distinct from that proposed for other class II aldolases is discussed. The results suggest tha t the class II aldolases should be sub-divided into two groups on the basis of both distinct folds and mechanism. (C) 1999 Academic Press.