Structure
Research ArticleThe three-dimensional structure of N -acetylneuraminate lyase from Escherichia coli
Introduction
N -acetylneuraminate lyase (EC 4.1.3.3) catalyzes the cleavage of N -acetylneuraminic acid (sialic acid, Neu5Ac) to pyruvate and N - acetyl- d -mannosamine (Figure 1). The enzyme plays an important role in the regulation of sialic acid metabolism in Escherichia coli [1]. Sialic acid is toxic to E. coli either as the free sugar or in the activated cytidine 5′ -monophosphorylated form [1]. Neu5Ac lyase is induced by sialic acid [2] and is able to regulate intracellular levels of the sugar. This control is as effective for sialic acid of biosynthetic origin as it is for exogenous sialic acid which may accumulate inside the bacterial cell [2]. Neu5Ac lyase has been found in pathogenic as well as non- pathogenic bacteria [3], [4], and in mammalian tissues [5].
Neu5Ac lyase also catalyzes the reverse aldol condensation reaction and has been used in that way to synthesize sialic acid, and some of its derivatives, from pyruvate and N -acetyl- d - mannosamine [6]. Interest in this aspect of the enzyme's activity has increased with the growing appreciation of the role of sialic acid in controlling biomolecular interactions, particularly at the surface of cells. An analogue of sialic acid is presently in the early stages of development as an anti-influenza virus compound [7].
The gene encoding E. coli Neu5Ac lyase has been cloned and over-expressed [6], [8], [9] and the enzyme has been biochemically characterized [3]. One of the reaction products, pyruvate, inhibits the enzyme at millimolar concentrations but N -acetyl- d - mannosamine does not [3], [9]. Sodium borotetrahydride inactivates the enzyme in the presence of either sialic acid or pyruvate [3], [9], suggesting that a Schiff base is formed during the enzyme reaction. The Neu5Ac lyase gene encodes a polypeptide of 297 amino acids [8] and several reports have suggested that the enzyme is a trimer of these polypeptides in solution [3], [8], [9]; this work shows the enzyme to be a tetramer.
Here we describe the crystallization of Neu5Ac lyase and its analysis by X-ray crystallography. The three-dimensional structure of the enzyme is compared with that of other aldolases of known three-dimensional structure. On the basis of sequence homology between Neu5Ac lyase, E. coli dihydrodipicolinate synthase (DHDPS) and the mos A gene product of Rhizobium meliloti, we anticipate that these proteins will have similar three- dimensional structures.
Section snippets
The structure of the protomer
The quality of the model presented here is described under Materials and methods. Neu5Ac lyase is folded as an α/β-barrel (Figure 2 and Figure 3), a structure first seen for triose phosphate isomerase [11] and subsequently observed in about 20 different enzymes [12] and a seed storage protein [13]. Neu5Ac lyase has a carboxy-terminal elaboration in the form of three additional α-helices. The secondary structural elements of the protein are recorded in Table 1. The β-barrel core of the structure
Biological implications
N -acetylneuraminate (Neu5Ac) lyase is a key enzyme in sialic acid catabolism in bacteria. In Escherichia coli, the protein efficiently regulates the intracellular concentration of sialic acid, preventing the accumulation of toxic levels of this sugar. The enzyme structure is shown here to be an α/β-barrel and its active site has been indirectly identified by analogy with other structures. Sequence identities of around 25 % with two other proteins, dihydrodipicolinate synthase and the mos A
Acknowledgements
We thank Drs Jose Varghese, Tom Garrett and Mark von Itzstein for helpful advice and discussions, Peter Hoyne for help with protein sequence data banks, Professor N Sakabe and the KEK National Laboratory for High Energy Physics for the use of Beam Line 6A2 at the Photon Factory, Neva Ivancic for over-expressing the enzyme, and Bert van Donkelaar for help with the X-ray data collection. Financial support of the Australian National Beamline Facility is acknowledged. TI thanks the Swiss National
Tina Izard, Biomolecular Research Institute, 343 Royal Parade, Parkville and School of Physics, The University of Melbourne, Parkville 3052, Australia.
Michael C Lawrence, Robyn L Malby, and Peter M Colman (corresponding author), Biomolecular Research Institute, 343 Royal Parade, Parkville.
Glenn G Lilley, CSIRO, Division of Biomolecular Engineering, 343 Royal Parade, Parkville 3052, Australia.
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Tina Izard, Biomolecular Research Institute, 343 Royal Parade, Parkville and School of Physics, The University of Melbourne, Parkville 3052, Australia.
Michael C Lawrence, Robyn L Malby, and Peter M Colman (corresponding author), Biomolecular Research Institute, 343 Royal Parade, Parkville.
Glenn G Lilley, CSIRO, Division of Biomolecular Engineering, 343 Royal Parade, Parkville 3052, Australia.