Heliobacter Urease
Created by Marina Piper
Ureases are hydrolases, using water to break urea into two molecules of ammonia (NH3) and one molecule of carbonic acid (H2CO3).1,2 Helicobacter mustelae synthesizes urease specifically to break down urea in the stomachs of ferrets (scientific name, Mustela putorius furo). By producing ammonia (a highly basic molecule), the bacterium is able to lower the pH in the surrounding vicinity, allowing the bacterium to survive and colonize.3 Attached to the mucosa of the ferret’s stomach, the bacterium causes inflammation (gastritis), and can lead to severe forms of gastroduodenal disease.3,4
The urease unique to Helicobacter mustelae is a dodecameric protein (from this point on denoted as UreA2B2; PDB entry 3QGA; see Image 1), sharing an identical quaternary structure with the urease belonging to Helicobacter pylori (UreAB; PDB entry 1E9Z; see Image 2).1 The basic subunit of both ureases is composed of a smaller peptide chain and a larger peptide chain, which then join together in groups of three to form trimers; four trimers make up the functional enzyme. Interactions between the chains are primarily the result of hydrogen and ionic bonds, though hydrophobic interactions do occur to a lesser extent. The overall structure is tetrahedral in nature and symmetrical, with a hollow center that fills with ammonia following the enzyme’s hydrolytic action.5
Composed of twelve subunits, the ureases found in the Helicobacter species are far larger than ureases found in bacteria of other genera. Klebsiella aerogenes also produces a urease (PDB entry 1EJW), which is also trimeric in nature, but is instead composed of an alpha, a beta and a gamma chain.6,7 The alpha chain is the largest of the three, and is analogous to the beta subunit H. pylori, and the beta 2 subunit in H. mustelae. (The nomenclature for the larger chain in the H. pylori urease is more variable, with some sources referring to it is as the alpha chain5, and others referring to it as the beta chain8; this paper will refer to it as the beta chain.) The Helicobacter equivalents of the beta and gamma chains of the K. aerogenes exist as a fusion peptide chain; in H. pylori this is simply denoted as the alpha chain8, and in H. mustelae it is (more descriptively) named “the fusion of urease gamma and beta subunits”1.
What distinguishes UreA2B2 structurally from UreAB is with regards to the metal ion it has bound at its active site. While most ureases discovered to date are nickel-bound enzymes, each beta 2 chain instead binds two iron cations.1 With two iron atoms per active site, one UreA2B2 molecule contains twenty-four iron atoms. This difference in ion-binding is attributed to the gastric environment of ferrets: nickel is far less available for the ureases to bind to than is iron (because of the carnivorous diet of ferrets), making the iron-containing form more successful in colonizing within the ferret’s stomach.1
Each subunit of the dodecameric protein contains an active site, for a total of twelve active sites for the entire protein. These are evenly spaced on the surface of the protein5, allowing urea to bind to the outside of the enzyme in order for it to carry out its catalytic function.
Each active site is composed of residues from the larger subunit (beta 2 or beta chains, for H. mustelae and H. pylori, respectively), with several of these residues being conserved between the two species’ ureases.1 Both UreA2B2 and UreAB’s active sites contain a lysine residue that has had a carboxyl group attached to the side-chain nitrogen (carbamylation); for UreA2B2, it is as position 218 of the beta 2 chain1, and for UreAB, it is at position 2195. The K. aerogenes urease has a carbamylated lysine at residue 2176, showing a high level of conservation between the different urease genes.
Carter et al., in studying the H. mustelae urease, created several mutant forms of the enzyme as a means of determining which residues were of significant importance to the catalytic site. The researchers replaced lysine 218 with three different residues: arginine, alanine and glutamic acid.1 As lysine is a basic amino acid, replacing it with a non-polar amino acid (such as alanine), or an acidic amino acid (such as glutamic acid) would not surprisingly lead to a reduction in catalytic ability. The ineffectiveness of the enzyme resulting from the switch from one basic amino acid (lysine) to another (arginine) not only shows the importance of having the carboxyl group attached to the residue, but also the high level of specificity that is necessary, such that the two additional nitrogens present in the arginine side chain will drastically affect the catalytic activity of this enzyme. This specificity explains why the carbamylated lysine residue is conserved in many, if not all, ureases.
Five other residues that contribute to active site formation and metal ion binding are also shared between H. mustelae and K. aerogenes. These include an aspartic acid (residue 360 for K. aerogenes; residue 361 for H. mustelae), four histidine residues (H134, H136, H246, and H272; H135, H137, H247, and H273).1 As with the carbamylated lysine residue, the obvious importance of the active site for urease function explains why these residues are so highly conserved across different urease-producing species, even to the point that they are all in nearly identical positions in the primary structure. The overall homology between various ureases is also fairly high, with 90% homology (with one gap) between the beta chain of H. pylori and the beta 2 of H. mustelae.8
Variation in the amino acid sequences of the different ureases does exist, however. The active site flap—which regulates substrate binding to and release from the active site7—has a conserved motif (helix-turn-helix) but varies between species with regards to the specific residues that make up this portion of the peptide.5 The difference in amino acid sequence not only leads to varying degrees in flexibility of the active site flap, but also helps explain the different affinities each enzyme has for its substrate (Km).5 Compared to K. aerogenes (2.3 mM)9, H. pylori has an extremely low Km value (0.17 mM)10.
While there is some level of variation in the amino acid sequence of the Helicobacter mustelae urease, UreA2B2, compared to other ureases, there is still a high degree of structural (and certainly functional) similarity between UreA2B2 and other ureases from different bacterial species and even different genera. As stated in the previous paper detailing the functional importance of the H. mustelae urease, these similarities could allow ferrets to serve as an animal model when studying the effects of H. pylori on humans. Though mice and humans share 99% of their DNA11, the genetic relationship between ferrets and humans, though as yet untested, could prove scientifically beneficial when it comes to research.