Abstract
The committed step of leucine biosynthesis, converting acetyl-CoA and α-ketoisovalerate into α-isopropylmalate, is catalyzed by α-isopropylmalate synthase (IPMS), an allosteric enzyme subjected to feedback inhibition by the end product L-leucine. We characterized the short form IPMS from Leptospira biflexa (LbIPMS2), which exhibits a catalytic activity comparable with that of the long form IPMS (LbIPMS1) and has a similar N-terminal domain followed by subdomain I and subdomain II but lacks the whole C-terminal regulatory domain. We found that partial deletion of the regulatory domain of LbIPMS1 resulted in a loss of about 50% of the catalytic activity; however, when the regulatory domain was deleted up to Arg-385, producing a protein that is almost equivalent to the intact LbIPMS2, about 90% of the activity was maintained. Moreover, in LbIPMS2 or LbIPMS1, further deletion of several residues from the C terminus of subdomain II significantly impaired or completely abolished the catalytic activity, respectively. These results define a complete and independently functional catalytic module of IPMS consisting of both the N-terminal domain and the two subdomains. Structural comparison of LbIPMS2 and the Mycobacterium tuberculosis IPMS revealed two different conformations of subdomain II that likely represent two substrate-binding states related to cooperative catalysis. The biochemical and structural analyses together with the previously published hydrogen-deuterium exchange data led us to propose a conformation transition mechanism for feedback inhibition mediated by subdomains I and II that might associated with alteration of the binding affinity toward acetyl-CoA.
Highlights
Isopropylmalate synthases (IPMSs) with and without a regulatory domain were found
We demonstrated that the activities of both LbIPMS1 and LbIPMS2 can be potentiated by several divalent metals with Mn2ϩ as the most effective activator followed by Mg2ϩ, Co2ϩ, and Ca2ϩ, whereas Zn2ϩ may slightly inhibit the activities of both enzymes (Fig. 2A)
We further tested a series of truncation mutants from the C terminus of subdomain II of LbIPMS2 to pinpoint the minimal structural components essential for catalysis (Fig. 5A)
Summary
Isopropylmalate synthases (IPMSs) with and without a regulatory domain were found. Results: IPMS subdomain II is essential for activities and likely involved in acetyl-CoA binding-mediated conformation transition. The biochemical and structural analyses together with the previously published hydrogen-deuterium exchange data led us to propose a conformation transition mechanism for feedback inhibition mediated by subdomains I and II that might associated with alteration of the binding affinity toward acetyl-CoA. LA_0469 was proved to encode an active IPMS with a significantly shorter peptide than those encoded by the other two genes (LA_2350, i.e. cimA and LA_2202, i.e. leuA1); it apparently lacks the regulatory domain and was designated as leuA2 This finding implied that the catalytic activity of the IPMS-like enzymes might be independent of the regulatory domain and directly led us to characterize the enzymatic features of both the long and short forms of IPMSs from Leptospira biflexa. Combining the previously published hydrogen-deuterium exchange data with our mutational analysis data, a potential mechanism involving acetyl-CoA binding mediated by the two subdomains and associated with acetyl-CoA binding is proposed for feedback inhibition
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
