Aspartate transcarbamoylase (ATCase; EC 188.8.131.52) catalyzes the second step of de novo
pyrimidine biosynthesis, the reaction between carbamoyl phosphate (CP) and aspartate to form N
-aspartate and inorganic phosphate (Jones et al.
), and is an important site of regulation of this pathway in many organisms. The structure and properties of the Escherichia coli
enzyme have been extensively studied (Herve, 1989
; Allewell, 1989
; Lipscomb, 1992
; England et al.
). It has a dodecameric structure (C6
) composed of two types of subunits. The two larger or catalytic subunits (C3
) are each composed of three identical polypeptide chains (M
33 000), while the three smaller or regulatory subunits (R2
) are each composed of two identical polypeptide chains (M
17 000). The catalytic chains contain two domains: the carbamoyl phosphate (CP) and aspartate-binding (ASP) domains. The regulatory chains also contain two domains: the nucleotide and Zn-binding domains. E. coli
ATCase is an allosteric enzyme that exhibits cooperativity for aspartate and heterotropic effects, being activated by ATP and inhibited by CTP. Upon binding the substrate aspartate (in the presence of saturating CP), E. coli
ATCase undergoes a conformational change from a low-activity T state to a higher activity R state. The large conformational differences in the X-ray structures of the unliganded ATCase (Stevens et al.
) and the N
-aspartate (PALA) liganded enzyme (Ke et al.
; Jin et al.
) have been proposed to define the structural differences between the T and R states.
Characterization of the ATCase enzyme from the hyperthermophilic and barophilic archaeon Methanococcus jannaschii
(Hack et al.
) suggested that it consists of catalytic trimers and regulatory dimers and has a molecular weight similar to that from E. coli
. Kinetic analysis of M. jannaschii
ATCase from cell-free extracts showed that it has limited homotropic cooperativity and little if any regulatory properties with ATP and CTP. Kinetic analysis of the M. jannaschii
catalytic trimer showed hyperbolic kinetics with an activation energy similar to that of the E. coli
trimer and with an activity that increases with temperature. It is stable at 358 K.
We have previously determined the structure of the catalytic trimer of M. jannaschii
ATCase in a monoclinic crystal form (Vitali et al.
). This study and comparisons with E. coli
ATCase and the hyperthermophilic ATCases from Pyrococcus abyssi
(Van Boxstael et al.
) and Sulfolobus acidocaldarius
(De Vos et al.
) provided insight into the strategies for thermostabilization adopted by the M. jannaschii
enzyme. We undertook the structural analysis of the orthorhombic form which is presented in this paper in order to further investigate the structure of the enzyme in different crystalline environments and its thermostabilization strategies. In particular, we were interested in the possible patterns of association of the catalytic subunits in the crystal. An interesting feature of the monoclinic form was the vertical association of two crystallographically independent catalytic subunits into a staggered dimer of trimers.