Structural characterization and comparison of the large subunits of IPM isomerase and homoaconitase fromMethanococcus jannaschii

Abstract

The aconitase family of proteins includes three classes of hydro-lyase enzymes: aconitases, homoaconitases and isopropylmalate (IPM) isomerases. They have a common Fe-S cluster-binding site and catalyze the isomerization of specific substrates by sequential dehydration and hydration. The archaeon Methanococcus jannaschii contains two aconitase family proteins, IPM isomerase and homoaconitase, which have 50% sequence identity. These two enzymes are heterodimeric proteins composed of large and small subunits encoded by separate genes. Although structures have been reported for the small subunits of the two enzymes, the first structures of oxidized and reduced forms of the large subunit of IPM isomerase (ox-MJ0499 and red-MJ0499, respectively) from M. jannaschii are reported here at 1.8 and 2.7 Å resolution, respectively, together with the structure of the large subunit of homoaconitase (MJ1003) at 2.5 Å resolution. The structures of both proteins have unbound Fe-S clusters and contain a fourth cysteine in the active site. The active site of MJ1003 is homologous to that of aconitase, whereas MJ0499 has significant structural distortion at the active site compared with aconitase. In addition, significant large conformational changes were observed in the active site of red-MJ0499 when compared with ox-MJ0499. The active sites of the two proteins adopt two different states before changing to the Fe-S cluster-bound `activated' state observed in aconitase. MJ1003 has an `open' active site, which forms an active pocket for the cluster, while ox-MJ0499 has a `closed' active site, with four cysteines in disulfide bonds. These data will be helpful in understanding the biochemical mechanism of clustering of the Fe-S protein family.

Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase

“…On the other hand, in the HACN subfamily, only enzymes from methanogenic archaea, including Methanococcus jannaschii , have the “fourth cysteine” at site 7 (light-green shadowed cysteine), which may be located near a sulfur atom in the [4Fe-4S] cluster; however, its role is currently unclear (Fig. 5a)4243. Indeed, we have already found that purified AcnX Type IIb protein (from Aeropyrum pernix K1) also contains iron ion(s), although functional annotation is in progress (unpublished).…”

“…( a ) Active site models of mAcn from bovine (PDB ID 1ACO)19 and ([Fe-S] cluster unbound) HACN of M. jannaschii (4KP2)42. So-called “Fe α ” is not ligated to a cysteine residue.…”

“…PaAcnX and AcnX from P. aeruginosa PAO1; AtAcnX, A. tumefaciens C58; TrAcnX, AcnX from T. reesei QM6a; BxAcnX, Burkholderia xenovorans LB400; ApAcnX, Aeropyrum pernix K1; AcnB from E. coli 18; mAcn from bovine19; AcnD from Shewanella oneidensis MR-121; IRP from human20; IPMI from Saccharomyces cerevisiae 26; HACN1 and HACN2 from M. jannaschii 2342; HACN3 from Pyrococcus horikoshii OT343. “S” and “L” indicate small and large subunits, respectively.…”

Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability

“…113 To date, only one structure of the large subunit of IPMI has been determined and reported. 114 The large subunit of Methanococcus jannaschii IPMI has been crystallized under aerobic conditions, and the structure has been determined at 1.8 Å resolution. 114 The monomer is composed of 18 α -helices and 17 β -strands distributed in three domains organized in a triangular manner.…”

Stereochemistry of enzymatic water addition to C = C bonds

“…The picture was made from PDB structure 4ADL, using PyMol (DeLano, 2002). Table 3 Hydration activity for fumaric acid and its derivatives with E-double bonds using porcine fumarase (Findeis and Whitesides, 1987;Teipel et al, 1968 (Lee et al, 2014). Since the important positively charged active site residues and the catalytic His101 and Ser264 are conserved in all enzymes, it is likely that the substrates will bind in a similar way.…”