Part:BBa_K1806006

Urease S. pasteruii

The natural urease of the bacteria specie S. pasteruii Urease catalyses the breakdown of urea to ammonia. This breakdown is a major endothermic process, conducting a high efficiency endothermic process.

A nickel-dependant enzyme[1], urease is effectively produced by many organisms using urea as a waste product.[2] Animals, plants, bacteria, fungi and some algs alike synthesize urease, which is also a present as a soil enzyme.[3] The breakdown of urea by urease is as follows:

(NH2)2CO + H2O+heat → CO2 + 2NH3 [4]

More specifically, urease catalyzes the hydrolysis of urea to produce ammonia and carbamate; the carbamate produced is subsequently degraded by spontaneous hydrolysis to produce another ammonia and carbonic acid.[5]

The significance of urease is that the reaction it catalyses has very high endothermic properties, meaning that there will be a significant heat intake in the conduction of the process, making the enzyme ideal to conduct a cooling process. Aside from its relatively high endothermic function, the enzyme has been historically important in major scientific establishments in respective fields and has been under the scope of many researchers worldwide, making urease one of the most identified enzymes in the world.[6]

Background Information

Urea and ureases have brought important turning points into modern biochemistry. Urea, which was first isolated from urine in 1773 was the first organic molecule to be synthesized from inorganic compounds in the laboratory by the works of Wöhler in 1828.

Following this breaking achievement, the year 1926 brought the studies of James B. Sumner, him purifying the the jackbean urease (Canavalia ensiformis) to be the first protein crystal ever obtained and his work was the study that was to prove that enzymes were proteins in structure. [7,8]

Urease was also the first enzyme to be purified. Being the enzyme to breakdown the major nitrogenous waste product, urea, the enzyme reached the glory of being the first enzyme to purified. It was with the purification of urease that it was understood that enzymes were functional outside of the cell. Also, many groundbreaking discoveries were made after this discovery, making urease one of the most commonly known and investigated enzymes of today. Since then, urease has been scrutinized in studies and has been described in terms of structure, activity, property and functionality. Urease was also the first enzyme to be crystallized by James B. Sumner in the year of 1926. It was his achievement that got him the Nobel Prize in 1946 and the world the understanding that enzymes were structurally proteins.

Structure and Activity of Urease

All ureases have functionally and structurally similar properties.[9] The structure complexes are not uniform, as there exist several chimeric structures, but in effect and complex activities the enzyme structures are have great similarity. The nickel-complex of all ureases are the main effector of the reaction complex.

The ureases that will be utilized in our project originate from the species of two different kingdoms, from the bacterium specie Sporosarcina pasteurii and the fungus specie Endocarpon pusillum. The functional efficiency and characterization of these two urease species are assumed to be different. There is an abundant foundation for research on S. pasteruii but the Endocarpon pusillum is an urease that remains to be characterized.

S. pasteruii is a trimeric enzyme with three chimeric parts, tagged as UreA, UreB and UreC. Almost all bacterial ureases have a similar trimeric enzyme structure and have very close activity levels.[8] These three subunits contain the α, ß and γ active sites that work in coordination to structurally change in the presence of urea to breakdown the molecule.[10] The effectivity of the S. pasteruii can be understood with the relative kinetic activity of the enzyme with its counterparts, the Soy-Bean Urease and Jack-Bean Urease. The kinetic degradation rate of SBU is 1/100 of S. pasteruii urease and this relative value is 1/14 for the JBU.[8] S. pasteruii urease has a significantly higher enzymatic kinetic activity in respect to other common ureases.

Also, urea is abundant substrate meaning that this reaction can reach high activity levels through high substrate concentration.

E. pusillum urease is a relatively newly discovered member of the urease family of enzymes. E. pusillum is a lichen-forming fungus species, making the expected structure of the urease similar to those of other fungal species.[11] The lack of characterization of the enzyme means that the initial characterization efforts may be being conducted in this study and that the primary data source may be found here.

Cloning

The sponsors providing the parts were not able to synthesize orders of more than 2000 bp. For this reason, the part was split into two respective segment parts to be ligated. The two segments were tagged as G-Block 10 and 11. Initially the G-Blocks were ligated to the pSB1C3 vector to be cloned. The verified cloning of the segment parts can be seen in the gel images below.

The two segment parts were ligated from the Hind3 restriction site. The ligated 10+11 urease complete part was cut with the EcoR1 and Pst1 restriction enzymes. The bands had the appropriate base lengths in the gel runs.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 206
  Illegal BamHI site found at 2
  Illegal XhoI site found at 2506
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 494
  Illegal NgoMIV site found at 876
  Illegal AgeI site found at 1673
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1629
  

References

1 Krajewska B, van Eldik R, Brindell M "Temperature- and pressure-dependent stopped-flow kinetic studies of jack bean urease. Implications for the catalytic mechanism". JBIC Journal of Biological Inorganic Chemistry 17 no.7(2012): 1123–1134. doi:10.1007/s00775-012-0926-8. PMC 3442171. PMID 22890689.

2 ZAMBELLI, BARBARA, FRANCESCO MUSIANI, STEFANO BENINI, and STEFANO CIURLI. "Chemistry of Ni2þ in Urease: Sensing, Trafficking, and Catalysis." 2011.

3 Ciurli S, Marzadori C, Benini S, Deiana S, Gessa C. “Urease from the soil bacterium Bacillus pasteurii: immobilization on Ca-polygalacturonate.” Soil Biol. Eiochem. 28 (1996): 81l-817.

4 "Chapter 16: Urease." In Helicobacter Pylori: Physiology and Genetics. Washington DC: ASM Press, 2001.

5 Zimmer M (Apr 2000). "Molecular mechanics evaluation of the proposed mechanisms for the degradation of urea by urease". J Biomol Struct Dyn. 17 (5): 787–97.doi:10.1080/07391102.2000.10506568. PMID 10798524.

6 Karplus, P. A., Pearson, M. A., & Hausinger, R. P. “70 years of crystalline urease: What have we learned?” Accounts of Chemical Research 30 no.8 (1997):330–337

7 Balasubramanian A, Ponnuraj K. “Crystal Structure of the First Plant Urease from Jack Bean: 83 Years of Journey from Its First Crystal to Molecular Structure.” J. Mol. Biol.400 (2010):274 – 283.

8 Zerner, B. “Recent advances in the chemistry of an old enzyme, urease.” Bioorg. Chem. 19 (1991):116-131

9 Follmer, Cristian, Rafael Real-Guerra, German E. Wasserman, Deiber Olivera-Severo, and Celia R. Carlini. "Jackbean, Soybean and Bacillus Pasteurii Ureases Biological Effects Unrelated to Ureolytic Activity." Eur. J. Biochem. 271 (2004): 1357-363. doi:10.1111/j.1432-1033.2004.04046.x.

10 Krajewska, Barbara. "Ureases I. Functional, catalytic and kinetic properties: A review". Journal of Molecular Catalysis B: Enzymatic 59 no.1-3 (2009):9–21. doi:10.1016/j.molcatb.2009.01.003

11 Sacristán M, Millanes A-M, Legaz M-E, Vicente C. “A Lichen Lectin Specifically Binds to the α-1,4-Polygalactoside Moiety of Urease Located in the Cell Wall of Homologous Algae.” Plant Signaling & Behavior.1 no.1(2006):23-27.