Active site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme.

The N-acetylneuraminate lyase (NAL) sub-family of (beta/alpha)(8) enzymes share a common catalytic step but catalyse reactions in different biological pathways. Known examples include NAL, dihydrodipicolinate synthetase (DHDPS), d-5-keto-4-deoxyglucarate dehydratase, 2-keto-3-deoxygluconate aldolase, trans-o-hydroxybenzylidenepyruvate hydrolase-aldolase and trans-2'-carboxybenzalpyruvate hydratase-aldolase. Little is known about the way in which the three-dimensional structure of the respective active sites are modulated across the sub-family to achieve cognate substrate recognition. We present here the structure of Haemophilus influenzae NAL determined by X-ray crystallography to a maximum resolution of 1.60 A, in native form and in complex with three substrate analogues (sialic acid alditol, 4-deoxy-sialic acid and 4-oxo-sialic acid). These structures reveal for the first time the mode of binding of the complete substrate in the NAL active site. On the basis of the above structures, that of substrate-complexed DHDPS and sequence comparison across the sub-family we are able to propose a unified model for active site modulation. The model is one of economy, allowing wherever appropriate the retention or relocation of residues associated with binding common substrate substituent groups. Our structures also suggest a role for the strictly conserved tyrosine residue found in all active sites of the sub-family, namely that it mediates proton abstraction by the alpha-keto acid carboxylate in a substrate-assisted catalytic reaction pathway.

Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase.

We describe here a sub-family of enzymes related both structurally and functionally to N-acetylneuraminate lyase. Two members of this family (N-acetylneuraminate lyase and dihydrodipicolinate synthase) have known three-dimensional structures and we now proceed to show their structural and functional relationship to two further proteins, trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and D-4-deoxy-5-oxoglucarate dehydratase. These enzymes are all thought to involve intermediate Schiff-base formation with their respective substrates. In order to understand the nature of this intermediate, we have determined the three-dimensional structure of N-acetylneuraminate lyase in complex with hydroxypyruvate (a product analogue) and in complex with one of its products (pyruvate). From these structures we deduce the presence of a closely similar Schiff-base forming motif in all members of the N-acetylneuraminate lyase sub-family. A fifth protein, MosA, is also confirmed to be a member of the sub-family although the involvement of an intermediate Schiff-base in its proposed reaction is unclear.

Investigation of the uptake of drugs by individual cells using a scanning proton microprobe (SPM).

In this paper we demonstrate the use of micro-PIXE (proton induced X-ray emission) for measuring the quantitative uptake of anti-AIDS drugs, containing metal atoms, by individual Vero cells (African green monkey kidney cell line). Hetero-polytungstates, which are assessed to present an activity against the HIV virus, were studied using Vero cells. It was found that unlike other techniques, SPM offers both the sensitivity and the spatial resolution to carry out these programs of investigations. The use of elemental analysis in single cells of cultured cell lines has shown to have distinct advantages over peripheral blood lymphocytes.

Structure of a dideoxynucleoside active against the HIV (AIDS) virus.

N4-Benzoyl-2',3'-dideoxycytidine, C16H17-N3O4.2H2O, M(r) = 351.4, monoclinic, P2(1), a = 4.691 (1), b = 14.448 (2), c = 12.924 (3) A, beta = 97.63 (1) degree, V = 868.2 (1) A3, Dm(flotation) = 1.34 (1), Dx = 1.344 Mg m-3, Z = 2, F(000) = 372, lambda = 1.5418 A, mu(Cu K alpha) = 0.65 mm-1, T = 292 (1) K, final R = 0.038 for 1437 observed data. The glycosidic torsion angle C(6)--N(1)--C(1')--O(4') is 20.6 (5) degrees and the pucker of the furanose ring is C(3') endo. Free rotation about the exocyclic C(4')--C(5') bond allows the hydroxymethyl substituent to adopt two orientations, trans and gauche, the latter resulting in a short contact, H(6)...O(5") of 2.21 (4) A, indicative of a relatively strong C--H...O intramolecular hydrogen-bonding interaction.