Methionine is an essential amino acid and plays an important role in a number of cellular functions, including the initiation of protein synthesis, biological methylation of DNA and rRNA and the biosynthesis of cysteine, phospholipids and polyamines. The biosynthesis of polyamines is involved in the control of many biological processes such as carcinogenesis, cell growth and differentiation. In the synthesis of the polymaines spermidine and spermine, methionine is consumed in a 1:1 stoichiometry with formation of the byproduct methylthioadenosine (MTA; Williams-Ashman & Canellakis, 1979
). Since the amount of methionine in cells is limited and de novo
synthesis of methionine is energetically costly, it is essential to salvage methionine and recycle the S atom from MTA (Trackman & Abeles, 1983
). Moreover, it has long been known that MTA, once converted to adenine and methylthioribose-1-phosphate (MTR-1-P; Ferro et al.
), is able to regenerate methionine (Williams-Ashman et al.
; Winans & Bassler, 2002
). This has been found in a wide variety of organisms, including mammals (Riscoe & Ferro, 1984
; Riscoe et al.
), Trypanosoma brucei
(Riscoe et al.
), Saccharomyces cerevisiae
(Subhi et al.
) and Klebsiella pneumoniae
(Furfine & Abeles, 1988
). The overall reaction scheme was determined in K. pneumoniae
(Furfine & Abeles, 1988
). However, the actual enzymes or genes involved in each step have not been unravelled.
A gene-inactivation study revealed that the S-box transcription-termination control system, first identified in Bacillus subtilis
, was used for regulation of gene expression in response to methionine availability (Grundy & Henkin, 1998
). The presence of the S-box motif provided the first indication that the mtnKA
genes could play a role in recycling MTA (Murphy et al.
). In 2003, we succeeded in showing the exact nature of five enzymes (MtnA, MtnW, MtnX, MtnB, MtnD) from B. subtilis
in the six steps of the downstream section of the methionine-salvage pathway (Ashida et al.
; Balakrishnan et al.
; Sekowska et al.
). The first step of the pathway in B. subtilis
is catalyzed by MtnA, which converts MTR-1-P into methylthioribulose-1-phosphate (MTRu-1-P).
The first ubiquitous enzyme of the pathway, MtnA, belongs to a family of proteins related to the α-subunit of eukaryotic translation initiation factor 2B (eIF2B), which is a heteropentameric protein composed of α–
subunits. Recently, the crystal structures of MtnA-homologous proteins [Ypr118w from S. cerevisiae
and an archaeal regulatory subunit (aIF2Bα) from Pyrococcus horikoshii
OT3] have been reported (Bumann et al.
; Kakuta et al.
). Despite structural analyses and genetic studies, the active site remains unclear. The primary structure and molecular weight of MtnA from B. subtilis
were very different from those of Ypr118w (38% identity, 45 kDa) and aIF2Bα (32% identity, 31 kDa). We have also established the generation of MTR-1-P, the substrate of MtnA, using recombinant B. subtilis
MtnK. For the purpose of investigation into the detailed catalysis of MtnA, the crystallization of MtnA complexed with MTR-1-P is currently in progress.
The methionine-salvage pathway is likely to be compartmentalized because of the extreme reactivity of the S atom towards dioxygen and radicals. Furthermore, the structure–function relationship of the series of B. subtilis enzymes remains unclear and their three-dimensional structures are unknown. Therefore, structural comparison between MtnA and its homologues will not only provide an evolutionary insight into two different catalytic mechanisms, but the three-dimensional structure of MtnA will also become an important first step in understanding the whole protein–protein network in the pathway. We report here the crystallization and the preliminary X-ray crystallographic study of MtnA from B. subtilis.