Sandbox Reserved 325
From Proteopedia
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==Structure== | ==Structure== | ||
<Structure load='2f6l' size='300' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_325/Chainbows/1' /> | <Structure load='2f6l' size='300' frame='true' align='left' caption='Insert caption here' scene='Sandbox_Reserved_325/Chainbows/1' /> | ||
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| - | Chorismate mutase is a homodimer which has an all-α-helical structure <ref name="pizza" />. There are 10 α-helicies spread across the two monomers of chorismate mutase <ref name="pizza" />. Aprozimately 86% of the amino acid residues are in the α-helicial formations <ref name="pizza" />. The α-helical structure of ''M. tuberculosis'' chorismate mustase similar to the chorismate mutases of ''S. cerevisae'' and ''E. coli''. It holds its dimeric state in a protein concentration as low as 5 nM <ref name="pizza" />. There are no β-sheets in chorismate mutase <ref name="CMArt2" /> | ||
| - | Chorismate has an active site, which is used for the catalysis of the shikimate pathway <ref name="pizza" />. The active site is made of Arg <sub>49</sub>,Lys <sub>60</sub>, Arg <sub>72</sub>, Thr <sub>105</sub>, Glu <sub>109</sub>, and Arg <sub>134</sub><ref name="pizza" />. This active site exists through electrostatic interactions with chorismate and hydrogen bonding between the amino acids <ref name="CMW2" />. The active site forms within a single chain <ref name="pizza" />. The active site can form without any help from the second half of the dimer <ref name="pizza" />. | ||
| - | The molecular weight of chorismate mutase is 36,000 Da <ref name="pizza" />. Based on the fact that each monomeric subunit has a molecular weight of 18,474 Da, the molecular weight of the molecule supports the theory that it is a dimer <ref name="pizza" />. This is also supported by the fact that all chorismate mutases that occur naturally are either trimers or dimers <ref name="pizza" /> | ||
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| - | not regulated by aromatic amino acids, which is supported by the fact that there are no allosteric regulatory sites. | ||
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| - | quaternary structure determined by molecular sieve chromatography | ||
| - | works best at 37 deg C ph 7.5 | ||
| - | 1 S-S bond between Cys 160 and cys 193 | ||
| - | aroQ | ||
| - | ph tolerance from 4.0 to 7.5 for optimal activity | ||
| - | no beta sheets 222222 | ||
| - | Mtb Chorismate Mutase Is a Dimeric Protein with a Predominantly | ||
| - | �-Helical Structure—Whereas catalytic activity and | ||
| - | regulatory activity of Mtb chorismate mutase point toward | ||
| - | some novel properties of the enzyme, the study was continued | ||
| - | to determine the biophysical parameters of the enzyme to define | ||
| - | the actual class to which it belongs. Size exclusion chromatography | ||
| - | was performed to determine the oligomeric state of | ||
| - | the protein. The output was a single peak corresponding to the | ||
| - | dimeric state of the recombinant protein (data not shown). In this | ||
| - | context, Mtb chorismate mutase is similar to the E. coli or yeast | ||
| - | chorismate mutases, which are also dimers of identical subunits | ||
| - | (24, 36). To determine the secondary structure of Mtb chorismate | ||
| - | mutase, the CD spectrum was recorded on a JASON spectropolarimeter | ||
| - | (Fig. 6). The data were analyzed using the K2D software | ||
| - | available on-line. The results suggest a predominantly | ||
| - | �-helical structure for the enzyme. This is reminiscent of the | ||
| - | AroQ class of enzymes from yeast and E. coli (24, 37) that are also | ||
| - | helical proteins. Members of the AroQ class of chorismate mutases | ||
| - | consist of unregulated and regulated (AroQr) enzymes and | ||
| - | are unusually divergent among closely related organisms (38). | ||
| - | This structure showed 71% helices with essentially no �-sheets. 2222222222 | ||
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here is a <scene name='Sandbox_Reserved_325/Disulfide/1'>disulfide bridge</scene> | here is a <scene name='Sandbox_Reserved_325/Disulfide/1'>disulfide bridge</scene> | ||
Revision as of 18:06, 3 April 2011
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| 2f6l, resolution 1.70Å () | |||||||||
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| Gene: | Rv1885c (Mycobacterium tuberculosis) | ||||||||
| Activity: | Chorismate mutase, with EC number 5.4.99.5 | ||||||||
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| Resources: | FirstGlance, OCA, RCSB, PDBsum | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
| This Sandbox is Reserved from January 10, 2010, through April 10, 2011 for use in BCMB 307-Proteins course taught by Andrea Gorrell at the University of Northern British Columbia, Prince George, BC, Canada. |
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Contents |
Chorismate Mutase
Introduction
The gene Rv1885c from Mycobacteria tuberculosis encodes for a non-functional chorismate mutase (*MtCM)[1]. This non-functional mutase has a 33-amino-acid cleavable sequence [1]. Chorismate mutase is a vital enzyme in the shikimate pathway, which allows for the synthesis of tryptophan, tyrosine, and phenylalanine [1]. This protein acts at the first branch point of the shikimate pathway, making it a regulating step in the conversion of prephenate from chorismate[2]. Chorismate mutase provides a 2x106 fold increase in the rate of reaction in comparision to the uncatalyzed reaction [3]. Chorismate mutase only occurs in bacteria, higher plants, and fungi, due to the fact that the shikimate pathway is only found in these organisms [4]. In Escherichia coli, chorismate mutase has a periplasmic destination[1]. In M. tuberculosis there is in abscence of a periplasmic compartment for chorismate mutase, so it secretes into the culture filtrate of M. tuberculosis[1]. It is believed that a pseudoperiplasmic space might exist in M. tuberculosis[1]. The N-terminal sequence of M. tuberculosis chorismate mutase is able to function in E. coli which suggests that M. tuberulosis chorismate mutase belongs to the AroQ class of the chorismate mutases[5]. Rv1885c is synthesized along with the 33-amino-acid terminal sequence, which when expressed with E. coli, is cleaved off the mature protein[1]. Chorismate mutase is the only example of an enzyme catalyzing a percyclic reaction [4]
Structure
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here is a
Mechanism
in michaelis menten kinetics it has Km of 0.5 ± 0.05 mM and Kcat of 60 s-1 Chorismate mutase is an essential enzyme in the shikimate pathway [1]. This pathway allows for the biosynthesis of aromatic amino acids tryptophan, tyrosine, and phenylalanine [1]. The production of tyrosine and phenylalanine is achieved by what is called a Claisen arrangement. first converting chorismate to prephenate. Prephenate then reacts with prephenate dehydratase and prephenate dehydrogenase which forms phenylpyruvate and hydroxyphenylpyruvate. After this occurs, aminotransferase converts hydroxy-phenylpyruvate and phenylpyruvate to phenylalanine and tyrosine. Chorismate mutase provides a 2x106 fold increase in the rate of reaction, in comparison to the uncatalyzed reaction [6]. It is the only example of an enzyme catalyzing a percyclic reaction [4]
Chorismate Mutase and Tuberculosis
may be involved in pathogenesis. one can take advantage of non-occurance of CMs in humans to try to develop antimicrobial drugs for human pathogens such as tb no func in non-shik pathways like those of macrophages of mammals. target this for TB infection ph is 4.5 om tb macrophage enviro. acidic.
Mycobacterium tuberculosis (Mtb)1 has developed ingenious mechanisms to survive inside the hostile environment presented by the host and to acquire essential nutrients from this adverse environment (1–3). The emergence of drug-resistant strains and synergy with the AIDS virus has further aggravated the disease scenario (4–6). For the development of new therapeutic intervention strategies, there is a need for identification of novel targets that are not only unique to Mtb but blocking of which would either prove lethal to the bacterium or render it extremely susceptible to the host immune response. In this context, understanding the mechanism of action of the aromatic amino acid pathway enzymes of Mtb assumes the utmost importance because most of the corresponding genes have been proven essential for the bacterium and have no human or mammalian counterpart (7, 8). Moreover, amino acid auxotrophs of Mtb do not survive or multiply in macrophages (9, 10), suggesting that these amino acids are not available within the compartment of the macrophage in which the bacteria reside. displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutase 2222222222222222222222222
References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Kim SK, Reddy SK, Nelson BC, Vasquez GB, Davis A, Howard AJ, Patterson S, Gilliland GL, Ladner JE, Reddy PT. Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids. J Bacteriol. 2006 Dec;188(24):8638-48. PMID:17146044 doi:188/24/8638
- ↑ PMID:PMC55368
- ↑ P.D. Lyne, A.J. Mulholland, W.G. Richards. Insights into chorismate mutase catalysis from a combined qm/mm simulation of the enzyme reaction. Journal of the American Chemistry Society. 1995 117(45):11345-11350
- ↑ 4.0 4.1 4.2 Kast P, Grisostomi C, Chen IA, Li S, Krengel U, Xue Y, Hilvert D. A strategically positioned cation is crucial for efficient catalysis by chorismate mutase. J Biol Chem. 2000 Nov 24;275(47):36832-8. PMID:10960481 doi:10.1074/jbc.M006351200
- ↑ Prakash P, Aruna B, Sardesai AA, Hasnain SE. Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity. J Biol Chem. 2005 May 20;280(20):19641-8. Epub 2005 Feb 28. PMID:15737998 doi:10.1074/jbc.M413026200
- ↑ P.D. Lyne, A.J. Mulholland, W.G. Richards. Insights into chorismate mutase catalysis from a combined qm/mm simulation of the enzyme reaction. Journal of the American Chemistry Society. 1995 117(45):11345-11350
