Abstract
Homoaconitase enzymes catalyze hydrolyase reactions in the alpha-aminoadipate pathway for lysine biosynthesis or the 2-oxosuberate pathway for methanogenic coenzyme B biosynthesis. Despite the homology of this iron-sulfur protein to aconitase, previously studied homoaconitases catalyze only the hydration of cis-homoaconitate to form homoisocitrate rather than the complete isomerization of homocitrate to homoisocitrate. The MJ1003 and MJ1271 proteins from the methanogen Methanocaldococcus jannaschii formed the first homoaconitase shown to catalyze both the dehydration of (R)-homocitrate to form cis-homoaconitate, and its hydration is shown to produce homoisocitrate. This heterotetrameric enzyme also used the analogous longer chain substrates cis-(homo)(2)aconitate, cis-(homo)(3)aconitate, and cis-(homo)(4)aconitate, all with similar specificities. A combination of the homoaconitase with the M. jannaschii homoisocitrate dehydrogenase catalyzed all of the isomerization and oxidative decarboxylation reactions required to form 2-oxoadipate, 2-oxopimelate, and 2-oxosuberate, completing three iterations of the 2-oxoacid elongation pathway. Methanogenic archaeal homoaconitases and fungal homoaconitases evolved in parallel in the aconitase superfamily. The archaeal homoaconitases share a common ancestor with isopropylmalate isomerases, and both enzymes catalyzed the hydration of the minimal substrate maleate to form d-malate. The variation in substrate specificity among these enzymes correlated with the amino acid sequences of a flexible loop in the small subunits.
Highlights
Attack of the coenzyme B (CoB) thiol on the methyl thioether of methyl-coenzyme M (CoM), releasing methane and forming a heterodisulfide compound (CoM-S-S-CoB) [1]
We previously identified one combination of M. jannaschii proteins (MJ0499 and MJ1277) as the isopropylmalate/citramalate isomerase that is involved in leucine and isoleucine biosyntheses [19]
By comparison with the homologous aconitase protein, the homoaconitase enzyme (HACN) enzyme is expected to catalyze the isomerization of homocitrate to homoisocitrate
Summary
Lyzed the hydration of cis-homoaconitate, cis-homo2aconitate, Hydrolyase Activity of the MJ1003/MJ1271 Proteins—The cis-homo3aconitate, and even the nonphysiological cis-. The MJ1003/MJ1271 any dehydratase activity observed in reactions containing 5 mM apoprotein was mixed with iron(II) and sulfide to reconstitute citrate or threo-DL-isocitrate. The resulting holoenzyme Steady-state kinetic parameters for HACN were calculated contained 5.1 Ϯ 0.2 iron equivalents per MJ1003/MJ1271 from the initial rates of the hydration reactions for all four dimer and 5.1 Ϯ 0.8 sulfide equivalents, characteristic of a sin- substrates (Table 1). To test whether this complex Michaelis constant is similar for all three physiologically relhad homoaconitase activity, cis-homoaconitate was synthe- evant substrates but is higher for cis-homo4aconitate. The fully recon- fore the methanogen HACN does not significantly discrimstituted MJ1003/MJ1271 holoenzyme catalyzed the hydration inate between substrates with 2–5 methylene groups in their of cis-homoaconitate with a specific activity of 0.8 unit mgϪ1 ␥-carboxylate chains.
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
