Crystal Structure of the Hexameric Catabolic Ornithine Transcarbamylase from Lactobacillus hilgardii: Structural Insights into the Oligomeric Assembly and Metal Binding

Abstract

Catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) catalyzes the formation of ornithine (ORN) and carbamoyl phosphate from citrulline, which constitutes the second step of the degradation of arginine via the arginine deiminase pathway. Here, we report the crystal structure of cOTC from the lactic acid bacteria Lactobacillus hilgardii (Lh-cOTC) refined to 2.1 A resolution. The structure reveals that Lh-cOTC forms a hexameric assembly, which was also confirmed by gel-filtration chromatography and analytical ultracentrifugation. The homohexamer, with 32 point group symmetry, represents a new oligomeric state within the members of the ornithine transcarbamylase family that are typically homotrimeric or homododecameric. The C-terminal end from each subunit constitutes a key structural element for the stabilization of the hexameric assembly in solution. Additionally, the structure reveals, for the first time in the ornithine transcarbamylase family, a metal-binding site located at the 3-fold molecular symmetry axis of each trimer.

New Insight into the Transcarbamylase Family: The Structure of Putrescine Transcarbamylase, a Key Catalyst for Fermentative Utilization of Agmatine

“…Another intriguing feature requiring functional clarification is our present finding of one Ni atom binding at the trimer threefold axis, at a site similar to the ones found in the catabolic OTCs from Lactobacillus hilgardii [16] and Giardia lamblia [22]. The finding of metals binding in these OTCs and the report that rat OTC is a conspicuous cadmium-binding liver protein [23] warrants the study of the significance of the metal interactions of transcarbamylases.…”

“…Supratrimeric architectures have been reported for pfOTC, which is a dodecameric tetramer of trimers (Figure 5B, leftmost panel) [21], and for the catabolic OTCs of Pseudomonas aeruginosa (again a dodecamer) [15] or L. hilgardii (a hexamer) [16] (Figure 5B, second and third panels from the left). Helix 1, the helix that is buried in PTC by helix 13, is centrally involved in the intertrimeric contacts that lead to the hexamer or dodecamers in these supratrimeric OTCs [15], [16], [21] (Figure 5B, details). We confirmed the involvement of helix 1 in the formation of the hexamers of PTC lacking helix 13 by crystallizing this truncated enzyme.…”

“…The Ni, which is octahedrally coordinated to three H69 N atoms (one per subunit) and three O atoms of fixed water molecules (Figure 4C), may have derived from the Ni-chelate column used for PTC purification. Ni was found binding in the same site of Lactobacillus hilgardii catabolic OTC [16]. Given the octahedral coordination that is characteristic for metals of the transition group II of the periodic table, including Cd [29], these observations of a metal site in PTC and in at least one OTC might perhaps explain the reported Cd avidity of liver OTC [23], rendering important to examine the significance of this metal site in these enzymes.…”

“…Sequence comparisons indicate that this helix, which has not been found in any other transcarbamylase, is constant among PTCs. We prove here by in silico studies and by helix deletion and experimental investigations (including X-ray crystallography of the truncated enzyme) that this C-terminal helix plays paramount roles in trimer stabilization and in the prevention of formation of supratrimeric oligomers similar to those seen with some OTCs [15], [16], [21]. This raises the question of which is the significance of higher oligomer formation among transcarbamylases.…”

“…Surprisingly, despite the fact that transcarbamylases catalyzing CP production from carbamyl group-containing compounds have been known for many years already, only one catabolic transcarbamylase, OTC, was studied in detail (see for example [15], [16]). Quite limited information, which does not include structural data, exists, for example, about PTC [7], [9], despite the fact that inhibition of agmatine catabolism in S. mutans might be of value in caries prevention [17].…”