is a Gram-positive and biotin-auxotroph bacterium that has long been used industrially to produce glutamate (Hermann, 2003
). Glutamate overproduction in C. glutamicum
is induced by the depletion of biotin (Shiio et al.
), the addition of a specific detergent such as polyoxyethylene sorbitan monopalmitate (Tween 40; Takinami et al.
) or the addition of a sublethal concentration of penicillin (Nunheimer et al.
). A recent study showed that the activity of the 2-oxoglutarate dehydrogenase complex (ODHC; EC 18.104.22.168) is reduced under each of these conditions (Shingu & Terui, 1971
; Kawahara et al.
), leading to an increase in the carbon flow towards glutamate synthesis at the ODHC branch point (Shimizu et al.
). However, the molecular mechanism underlying glutamate overproduction remains elusive.
was identified as a suppresser gene for a Tween 40-sensitive mutant strain that is more sensitive to Tween 40 for glutamate production than the wild type (Kimura et al.
). Genetic and biochemical studies showed that dtsR1
encodes the carboxyltransferase subunit (CT) of the acetyl-CoA carboxylase complex (ACC; EC 22.214.171.124), which catalyzes the first committed step in fatty-acid biosynthesis (Kimura et al.
; Gande et al.
). It was observed that disruption of dtsR1
causes a reduction in ODHC activity, resulting in constitutive glutamate production in the absence of inducers (Kimura et al.
). In addition, overexpression of dtsR1
inhibits glutamate production (Kimura et al.
). These studies suggested that a functional (and probably physical) interaction between DtsR1 and ODHC is involved in glutamate production and that a reduction in DtsR1 activity triggers a reduction in ODHC activity, enhancing the glutamate synthesis from 2-oxoglutarate (Kimura et al.
). Therefore, DtsR1 is considered as a potential target for metabolic engineering to improve glutamate overproduction in C. glutamicum
. Towards this end, knowledge of the structure–function relationship of DtsR1 is required.
Several crystal structures of bacterial CT homologues and eukaryotic CT domains have already been determined: those of transcarboxylase 12S from Propionibacterium shermanii
(Hall et al.
), a CT of the sodium ion pump glutaconyl-CoA decarboxylase from Acidaminococcus fermentans
(Gcdα; Wendt et al.
), a CT of propionyl-CoA carboxylase from Streptomyces coelicolor
PCCB; Diacovich et al.
), a CT of acyl-CoA carboxylase from Mycobacterium tuberculosis
AccD5; Lin et al.
), the CTs of ACC from Staphylococcus aureus
CT) and Escherichia coli
CT; Bilder et al.
) and a CT domain of ACC from Saccharomyces cerevisiae
(yeast-CT; Zhang et al.
). The typical CT subunit is composed of two repetitions of a crotonase-fold domain. Although the structures of these CTs are similar to one another, with root-mean-square deviations in the range 0.79–1.91 Å, the surface residues of these CTs, which include the substrate-binding site and its peripheral region, are not well conserved. These differences seem to be important for substrate-specificity and interaction with specific biotinoyl carboxyl carrier proteins. DtsR1 shows 48.4, 20.9, 57.8, 65.5, 19.1, 20.1 and 20.3% amino-acid identity with 12S, Gcdα, Sc
CT and yeast-CT, respectively, suggesting that DtsR1 has essentially the same fold as related proteins. However, since the differences between DtsR1 and other related CTs, most of which are expected to be located on the surface of the DtsR1 molecule, are likely to be responsible for the substrate-specificity and species-specific interaction with biotinoyl carboxyl carrier protein, detailed structural information is still required in order to understand the structure–function relationship of DtsR1. Therefore, we initiated structural analysis of DtsR1 in order to understand the details of its structure–function relationship for use in metabolic engineering of C. glutamicum
. Here, we report the overexpression, crystallization and preliminary crystallographic analysis of DtsR1.