Methanogen Homoaconitase Catalyzes Both Hydrolyase Reactions in Coenzyme B Biosynthesis

Abstract

Homoaconitase enzymes catalyze hydrolyase reactions in the ␣-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 cishomoaconitate, 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.

Substrate Specificity Determinants of the Methanogen Homoaconitase Enzyme: Structure and Function of the Small Subunit,

“…Expression was then induced by the addition of 50 μM isopropyl 1-β-D-galactopyranoside, and the culture was then incubated for an additional 3-4 h. The wild-type MJ1003 and MJ1271 variant proteins were purified by heating the cell lysate, followed by anion-exchange chromatography, dialysis, and concentration by centrifugal ultrafiltration (1). Protein purity was determined by SDS-PAGE with Coomassie Blue dye staining.…”

“…For cis-aconitic acid: 1 A recently purchased batch of cisaconitic acid (Sigma) contained 7% trans-aconitic acid. A fresh sample of the latter batch was dissolved in water, immediately adjusted to pH 7 using NaOH, stored briefly on ice, and used for the kinetic experiments reported here.…”

“…These two protein sequences are 53% identical, and both were previously annotated as IPMI subunits based on primary sequence alone, only to be distinguished by heterologous expression, purification, reconstitution, and in vitro kinetic analysis (1,11). Sequences of the predicted flexible loop regions of these enzymes, identified by homology models and sequence alignments, may be the best indicators of substrate specificity.…”

Archaeal ApbC/Nbp35 Homologs Function as Iron-Sulfur Cluster Carrier Proteins

“…FeCl 3 was added to the stirred reaction mixtures at 0°C to obtain a concentration of 500 M. After 10 min, Na 2 S was added dropwise to reach a concentration of 500 M, and the mixture was stirred under argon for 2 h to reconstitute clusters in holoproteins. The MMP0704 holoproteins were desalted using a PD-10 column (GE Healthcare) equilibrated with an anoxic solution of 50 mM Tris-HCl (pH 8.0) (12). Iron and sulfide analyses were Complementation vector 2 performed using standard methods (3,41).…”

Automated interpretation of metabolic capacity from genome and metagenome sequences

“…The reaction module RM001 corresponds to KEGG modules M00010 (observed in 72% of 2800 genomes), M00433 (4.6%), M00608 (1.7%), M00535 (4.6%), and M00432 (69%), which share paralogous gene sets [8,9]. M00606 defined as (K10977 K16792 + K16793 K10978) is for chain extensions from 2-oxoglutarate to 2-oxoadipate and further to 2-oxosuberate in methanogenic archaea, which are apparently catalyzed by the same set of enzymes [13][14][15]. According to our annotation, which can be examined from the Ortholog table link of M00606, the synthase K10977 (aksA) is limited to methanogenic archaea, but the dehydratase complex, K16792 (aksD) and K16793 (aksE), is also found in Chloroflexi (green non-sulfur bacteria) and Deinococcus-Thermus.…”