Methionine is an essential amino acid for cellular functions such as protein synthesis (Cooper, 1983
), transmethylation (Cantoni, 1951
) and polyamine biosynthesis (Stipanuk, 1986
; Tabor & Tabor, 1976
). Bacteria and eukarya usually possess the methionine-salvage pathway (MSP; Sekowska et al.
), which salvages methionine from methylthioadenosine, the end product of spermidine and spermine anabolism (Winans & Bassler, 2002
). The MSP was first unravelled in Bacillus subtilis
(Ashida et al.
; Sekowska & Danchin, 2002
) and functional MSPs have been experimentally demonstrated for various organisms (Pirkov et al.
; Sekowska et al.
; Sufrin et al.
). At least seven enzymes are involved in the MSP of B. subtilis
. Of these enzymes, 2,3-diketo-5-methylthiopentyl-1-phosphate enolase (DK-MTP-1P enolase) catalyzes the enolization of 2,3-diketo-5-methylthiopentyl-1-phosphate.
DK-MTP-1P enolase has attracted enormous attention owing to its homology to the large subunit of d
-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). RuBisCO plays a key role in fixing CO2
into a five-carbon sugar phosphate in the Calvin cycle (Lorimer, 1981
). However, RuBisCO is an inefficient catalyst; therefore, the development of a super-RuBisCO is a potential avenue to improve crop productivity. Phylogenetic analyses have indicated that RuBisCOs can be divided into four forms (forms I–IV) and DK-MTP-1P enolase belongs to form IV (Tabita, 1999
; Tabita et al.
). Because the form IV proteins do not catalyze either carboxylation or oxygenation, they are called ‘RuBisCO-like proteins’ (RLPs). Based on their quaternary structures, different forms of RuBisCO can be also distinguished. In forms I and II a hexadecameric (L8
) form is found that consists of eight 55 kDa large (L) subunits and eight 15 kDa small (S) subunits. A dimeric (L2
) enzyme, similar in structure to the L subunits of the hexadecameric enzymes, is restricted to some form III RuBisCOs and RLPs.
The activation and reaction of RuBisCO involve multiple discrete steps. In activation, RuBisCO is carbamylated by the addition of an activator CO2
followed by binding of Mg2+
. In carboxylation, the substrate, d
-ribulose-1,5-bisphosphate, binds to the carbamylated active site; this is followed by the binding of gaseous CO2
to yield two molecules of 3-phosphoglycerate as products (Andrews & Lorimer, 1987
; Hartman & Harpel, 1994
). Structural analyses have clarified the RuBisCO structures in these multiple steps, which include apo decarbamylated (E), apo carbamylated (ECM), holo decarbamylated (E-ligand) and holo carbamylated (ECM-ligand) forms (Andersson & Backlund, 2008
; Schneider et al.
; Spreitzer & Salvucci, 2002
). Depending on the activation and ligand-binding states, two flexible catalytic loops, called loop-6 and the 60s loop, are either ‘open’ or ‘closed’ to adjust the degree of solvent accessibility to the active site (Schreuder et al.
DK-MTP-1P enolase is considered to partially share the same activation mechanism as RuBisCO. On the basis of structural and biochemical studies (Imker et al.
), the enzyme from Geobacillus kaustophilus
is activated via
carbamylation of Lys173 at the active site and subsequent coordination by Mg2+
. Therefore, the structure of DK-MTP-1P enolase must exhibit multiple states including E, ECM, E-ligand and ECM-ligand forms, as demonstrated by structures of RuBisCO. At present, crystal structures of DK-MTP-1P enolase are not available in all forms. The structure of the apo decarbamylated form (E) remains unknown, although crystal structures of DK-MTP-1P enolase from G. kaustophilus
(60% sequence identity to the B. subtilis
enzyme) have been reported in four forms (Imker et al.
): a PO4
-bound decarbamylated (unactivated) form (E-PO4
), a carbamylated (activated) form complexed with Mg2+
(ECM), a carbamylated form complexed with Mg2+
and bicarbonate (ECM-HCO3
) and a carbamylated form complexed with Mg2+
and the alternate substrate 2,3-diketohexane-1-phosphate (ECM-DK H-1P). If the structure of the E form of DK-MTP-1P enolase were available, the dynamic events that occur in the discrete catalytic steps of DK-MTP-1P enolase could be elucidated. It is also possible to compare the structures with those of RuBisCO in the discrete catalytic steps, which might provide clues about the functional and evolutionary relationships between RLP and RuBisCO. Furthermore, we can compare the E-form structures of DK-MTP-1P enolase and other RLPs from Chlorobium tepidum
(26% sequence identity to the B. subtilis
enzyme; Li et al.
) and Rhodopseudomonas palustris
(25% sequence identity to the B. subtilis
enzyme; Tabita et al.
), the molecular functions of which remain unknown. Such comparisons may provide further insights into the molecular mechanisms of RLPs of currently unknown function.
In order to understand the structure–function relationship, crystallization of the apo decarbamylated form (E) of DK-MTP-1P enolase from B. subtilis (molecular weight 45.1 kDa) has been attempted. Here, we report the purification, crystallization and preliminary X-ray crystallographic study of B. subtilis DK-MTP-1P enolase.