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In JoVE (1)
Other Publications (3)
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Articles by Usheer Kanjee in JoVE
Test för att mäta aktiviteten hos Escherichia coli Inducible Lysin Decarboxyase
Usheer Kanjee, Walid A. Houry
Department of Biochemistry, University of Toronto
Aktiviteten i den inducerbara lysin decarboxylase övervakas genom att reagera substratet L-lysin och produkten cadaverine med 2,4,6-trinitrobenzensulfonic syra och bildar addukter som har differentiell löslighet i toluen.
Other articles by Usheer Kanjee on PubMed
Structure of RavA MoxR AAA+ Protein Reveals the Design Principles of a Molecular Cage Modulating the Inducible Lysine Decarboxylase Activity
Proceedings of the National Academy of Sciences of the United States of America. Dec, 2010 | Pubmed ID: 21148420
The MoxR family of AAA+ ATPases is widespread throughout bacteria and archaea but remains poorly characterized. We recently found that the Escherichia coli MoxR protein, RavA (Regulatory ATPase variant A), tightly interacts with the inducible lysine decarboxylase, LdcI/CadA, to form a unique cage-like structure. Here, we present the X-ray structure of RavA and show that the αβα and all-α subdomains in the RavA AAA+ module are arranged as in magnesium chelatases rather than as in classical AAA+ proteins. RavA structure also contains a discontinuous triple-helical domain as well as a β-barrel-like domain forming a unique fold, which we termed the LARA domain. The LARA domain was found to mediate the interaction between RavA and LdcI. The RavA structure provides insights into how five RavA hexamers interact with two LdcI decamers to form the RavA-LdcI cage-like structure.
Linkage Between the Bacterial Acid Stress and Stringent Responses: the Structure of the Inducible Lysine Decarboxylase
The EMBO Journal. Mar, 2011 | Pubmed ID: 21278708
The Escherichia coli inducible lysine decarboxylase, LdcI/CadA, together with the inner-membrane lysine-cadaverine antiporter, CadB, provide cells with protection against mild acidic conditions (pH∼5). To gain a better understanding of the molecular processes underlying the acid stress response, the X-ray crystal structure of LdcI was determined. The structure revealed that the protein is an oligomer of five dimers that associate to form a decamer. Surprisingly, LdcI was found to co-crystallize with the stringent response effector molecule ppGpp, also known as the alarmone, with 10 ppGpp molecules in the decamer. ppGpp is known to mediate the stringent response, which occurs in response to nutrient deprivation. The alarmone strongly inhibited LdcI enzymatic activity. This inhibition is important for modulating the consumption of lysine in cells during acid stress under nutrient limiting conditions. Hence, our data provide direct evidence for a link between the bacterial acid stress and stringent responses.
The Enzymatic Activities of the Escherichia Coli Basic Aliphatic Amino Acid Decarboxylases Exhibit a PH Zone of Inhibition
Biochemistry. Nov, 2011 | Pubmed ID: 21957966
The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH.