Hydroxyproline (trans-4-hydroxy-L-proline (4-L-Hyp)) can be used by certain microorganisms as a source of carbon and nitrogen. The nitrogen fixing bacterium, Sinorhizobium meliloti carries a cluster (hyp cluster) of 14 genes responsible for the transport and degradation of this amino acid in the cell. The biological functions of several gene products in the hyp cluster are still unknown. So far, it is known that the conversion of trans-4-hydroxy-proline to α-ketoglutarate, one of the intermediate of the TCA cycle, occurs in four enzymatic reactions.
The whole hyp cluster is up regulated in the presence of 4-hydroxy-proline in the media. Previous studies have shown several other 4-hydroxy-proline-inducible genes. One of these genes, smc04388, has been annotated as a putative omega amino transaminase. The role of this omega transaminase in the main catabolic pathway of 4-hydroxy-proline has not been investigated. In order to address this, two mutant strains; a single smc04388 mutant and a double smc04388 hypD mutant were created. Growth curves of these mutants in minimal media showed that the Smc04388 protein is not required for the growth of S. meliloti in the presence of trans-4-hydroxy-L-proline as the sole carbon and nitrogen source.
The Smc04388 protein was overexpressed as a Strep-tagged and purified from S. meliloti. The purified enzyme showed amino transaminase activity with pyruvate and α-methylbenzylamine. In addition, an enzymatic reaction using the product of the second enzyme of the 4-hydroxy-proline pathway, Δ1-pyrroline-4-hydroxy-2-carboxylate, was carried out to test the activity of Smc04388 with this compound. Mass spectrometry analysis of this reaction mixture revealed the formation of L-alanine from pyruvate and Δ1-pyrroline-4-hydroxy-2-carboxylate, suggesting the utilization of this compound as an amino donor by the Smc04388 transaminase. It was also shown that transamination activity in cell extracts increase in the absence of Δ1-pyrroline-4-hydroxy-2-carboxylate deaminase, the enzyme that catalyzes the conversion of this compound in the 4-hydroxy-proline pathway. These results confirmed the hypothesis that the Smc04388 omega amino transaminase is involved in a secondary pathway related to the known catabolic pathway of 4-hydroxy-proline in bacteria. Further understanding of this secondary pathway will contribute to the study of the metabolism of 4-hydroxy-proline in bacteria. In addition to this, the complete characterization of the Smc04388 omega amino transaminase could have practical application in the pharmaceutical industry. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/15463 |
| Date | January 2014 |
| Creators | Perez, Hernandez Guianeya |
| Contributors | Finan, Turlough, Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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