Urease crystal structure

The crystal structure of urease form Klebsiella aerogenes has recently been determined (47). The two nickel(II) ions in the active site are... 

Urease (urea amidohydrolase) is an enzyme first identified over a hundred years ago in bacterial extracts [22], The presence of urease is a virulence factor for some pathogenic bacteria [23,24], It is now known to occur also in plants, fungi, and invertebrates (see [24,25] for reviews). Urease from jack bean was the first enzyme to be crystallized, in 1926. Almost 50 years later its metal content was reexamined and it was found to contain two atoms of nickel per subunit [26]. Finally in 1995 the crystal structure of the enzyme from the enteric bacterium Klebsiella aerogenes was determined [27], Amino-acid sequence comparisons predict that the structures of the plant and bacterial enzymes are similar, although with different subunit arrangements. 

A clearer picture emerged when the crystal structure of K. pneumoniae urease was determined [27], The nickel atoms in the center, Ni-1 and Ni-2, are 3.5 A apart. They are bridged by a carbamyl group, formed from C02 and a lysine residue, explaining the requirement for hydrogen carbonate in reconstitution. The other ligands are two histidines for Ni-1 and an aspartate and two histidines for Ni-2. 

From the crystal structure of urease, Jabri et al. [27] proposed that urea binds through its carbonyl oxygen, whereas the -NH2 hydrons are hydrogen-bonded to residues in the protein (Figure 1). The structure of the site is such that water molecules in the active site do not coordinate optimally to the nickel ions in the substrate-free form. As a result, the binding of urea is favored [40], A loop of polypeptide forms a flap that covers the active site once urea is bound. This flap includes cysteine 319, which had been believed to be catalytically important [41] and is one of the residues proposed to hydrogen-bond to the urea nitrogens. Mutation of this cysteine to alanine leads to decrease, but not necessarily loss, of activity. 

Jabri E et al (1995) The crystal structure of urease from Klebsiella aerogenes. Science 268 998-1004 PDBID 2KAU... 

Metalloenzymes pose a particular problem to both experimentalists and modelers. Crystal structures of metalloenzymes typically reveal only one state of the active site and the state obtained frequently depends on the crystallization conditions. In some cases, states probably not relevant to any aspect of the mechanism have been obtained, and in many cases it may not be possible to obtain states of interest, simply because they are too reactive. This is where molecular modeling can make a unique contribution and a recent study of urease provides a good example of what can be achieved119 1. A molecular mechanics study of urease as crystallized revealed that a water molecule was probably missing from the refined crystal structure. A conformational search of the active site geometry with the natural substrate, urea, bound led to the determination of a consensus binding model[I91]. Clearly, the urea complex cannot be crystallized because of the rate at which the urea is broken down to ammonia and, therefore, modeling approaches such as this represent a real contribution to the study of metalloenzymes. 

Historically, the first chemical synthesis of urea by Wohler, from ammonium cyanate in 1828, was a milestone that established a bridge between inorganic and organic chemistry. Urease was the first enzyme ever to be crystallized (6), and it was the first protein shown to contain nickel ions in the active site (7). The first X-ray crystal structure of urease became known in 1995 (8). Significant progress was made since then toward an understanding of its catalytic mechanism, as well as toward the structural and functional emulation of its active site by synthetic model complexes (5, 9). 

An alternative scenario was put forward based on the crystal structures of urease inhibited by either phosphate, diamidophosphate, or borate (4,5, 28). It gets some support from the kinetic findings for fluoride inhibition of urease (29), as well as from recent model calculations (30, 31). Boric acid, known to be a competitive inhibitor of urease, can be considered a good substrate analogue, since it is isoelectronic with urea and has the same shape and dimension. Bacillus pasteurii could be crystallized in the presence of boric acid. The structure reveals that a molecule of B(OH)3 is symmetrically spanning the nickel ions, replacing Wj, W2,... 

Without doubt, ligands based on bridging alkoxide or phenolate groups are most prominent in biomimetic or bioinspired chemistry with bimetallic sites. Even before the X-ray crystal structure of urease became known, first model compounds using this ligand type were developed. Complex 3, reported in 1989 by Buchanan,... 

The nickel enzymes covered in this article can be divided into two groups redox enzymes and hydrolases. The five Ni redox enzymes are hydrogenase, CO dehydrogenase (CODH), acetyl-CoA synthase (ACS), methyl-Coenzyme M reductase (MCR), and superoxide dismutase (SOD). Glyoxalase-I and urease are Ni hydrolases. Ni proteins that are not enzymes are not covered, because they have been recently reviewed. These include regulatory proteins (NikR) and chaperonins and metal uptake proteins (CooJ, CooE, UreE, and ABC transporters). A recent crystal structure of NikR, shown in Figure l(i), is a notable recent achievement in this area. ... 

The NagA enzyme from E. coli, unambiguously a member of CE 9, however, on isolation was found to contain one Zn " per active site and no Fe and was powerfully inhibited (K = 34 nM) by the methyl phosphoramidate analogue of the substrate, which accurately mimics the tetrahedral intermediate in both carboxypeptidase- and urease-like mechanisms. The metal dependence of CE 9 appears variable and both urease-like mechanisms and mechanisms analogous to that of CE 4 may operate. The crystal structure of the E. coli enzyme reveals only bound Zn, in a site equivalent to one of the two Fe sites of the B. subtilis enzyme and no potential histidine ligands for the other Fe the T. maritime enzyme appears to be intermediate, with one Fe bound. 

Table 1 summarizes the general characteristics of representative urease, hydrogenase and CODHs. As it will be further discussed below, the X-ray structures of only two Ni-containing proteins, urease and hydrogenase, are known [16, 17]. The former has the well known triose phosphate isomerase (TIM) barrel topology (Fig. 1) whereas the latter displays a so far unique folding (Fig. 2). The next challenge will be the elucidation of the crystal structures of the CODH/ACS enzyme of Clostridium thermoaceticum and of the simpler CODH from Rhodospirillum rubrum. 

Song HK, Muleooney SB, Hubee R and Hau-siNGEE RP (2001) Crystal structure of Klebsiella aerogenes UreE, a nickel-binding metallochaperone for urease activation. J Biol Chem 276 49359-49364. 

High resolution structures of urease from B. pasteurii complexed with /3-mercaptoethanol (13) (BME Fig. IB) and acetohydroxamic acid (14) (AHA Fig. 1C) indicate the presence of bridging/chelating coordination to the Ni ions, highlighting the importance of both metal ions in the reactivity of the enzyme. The structures of B. pasteurii urease crystallized in the presence of phosphate... 

Prior to the crystallization of jack bean urease it was assumed by the biochemical community that enzymes had no ordered structure. In 1965 the first crystallographic evidence for the mechanism by which enzymes work when Phillips and his group solved the lysozyme structure [6], Details of the structure indicated how the enzyme could bind the oligosaccharides present in its target, bacterial cell wall peptidoglycans, and could respond to the binding event by changing its structure. 

Buchners fermentation of sugar from yeast extracts Sumner s crystallization of urease Flemming s discovery of chromosomes Mendel s characterization of genes Miescher s isolation of nucleic acids Watson and Crick s structure of DNA... 

A for the two histidines. The other two Cu" ions are found in surface exposed sites coordinated by two histidine e nitrogens and one or two water molecules. The putative cysteine ligand identified by spectroscopy and mutagenesis " does not bind Cu" in the structure and is quite distant from the metal-binding sites. It may be that this interaction occurs in solution between a cysteine residue from one dimer and a Cu" ion from a second dimer, and is precluded in the structure by crystal packing. The dimer interface site is proposed to deliver Ni" ions one at a time to the urease active site, and the other two sites are proposed to play a more secondary role, serving as reservoirs for Ni". ... 

Enzymes that hydrolyze proteins and other compounds composed of amino acids were among the first biological catalysts to be discovered, and they have continued to be prominent in studies of enzyme structure, kinetics, activation, and mechanism of action. The crystallization of the enzyme urease by Sumner was followed by the crystallization of various proteolytic enzymes in the laboratory of Northrop. These studies established that catalytic activity is associated with what appear to be pure proteins all well-defined enzymes isolated subsequently have also proved to be proteins, although many contain additional components. The study of enzymatic reactions involving proteins as substrate, therefore, gives insight into the chemical nature of enzymes as well as the mechanisms by which they act. 

There is no doubt that as with all science there will be an element of luck involved. In this case part of it was the chance meeting of individuals who had a common interest This was followed by a bit of careful discussion and extra experiments. Some proteins will crystdlise out from crude solutions, urease is a classic example, but there is no doubt that high protein purity and careful handling, especially in respect of potential sources of proteolysis, definitely improve the chances of successful crystallization. If however you want the structure badly enough then you should also be prepared to obtain the enzyme from several different sources as there is no predicting which source will turn up trumps ... 

---