A single mutation in the active site swaps the substrate specificity of N-acetyl-L-ornithine transcarbamylase and N-succinyl-L-ornithine transcarbamylase

Abstract

Transcarbamylases catalyze the transfer of the carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate such as aspartate, ornithine, or putrescine. Previously, structural determination of a transcarbamylase from Xanthomonas campestris led to the discovery of a novel N-acetylornithine transcarbamylase (AOTCase) that catalyzes the carbamylation of N-acetylornithine. Recently, a novel N-succinylornithine transcarbamylase (SOTCase) from Bacteroides fragilis was identified. Structural comparisons of AOTCase from X. campestris and SOTCase from B. fragilis revealed that residue Glu92 (X. campestris numbering) plays a critical role in distinguishing AOTCase from SOTCase. Enzymatic assays of E92P, E92S, E92V, and E92A mutants of AOTCase demonstrate that each of these mutations converts the AOTCase to an SOTCase. Similarly, the P90E mutation in B. fragilis SOTCase (equivalent to E92 in X. campestris AOTCase) converts the SOTCase to AOTCase. Hence, a single amino acid substitution is sufficient to swap the substrate specificities of AOTCase and SOTCase. X-ray crystal structures of these mutants in complexes with CP and N-acetyl-L-norvaline (an analog of N-acetyl-L-ornithine) or N-succinyl-L-norvaline (an analog of N-succinyl-L-ornithine) substantiate this conversion. In addition to Glu92 (X. campestris numbering), other residues such as Asn185 and Lys30 in AOTCase, which are involved in binding substrates through bridging water molecules, help to define the substrate specificity of AOTCase. These results provide the correct annotation (AOTCase or SOTCase) for a set of the transcarbamylase-like proteins that have been erroneously annotated as ornithine transcarbamylase (OTCase, EC 2.1.3.3).

Figure 1.

N-acetylnorvaline or N-succinylnorvaline binding of SOTCase or AOTCase mutants. (A–D) Contours of the electron density map (2F_o − F_c) (1.0 σ shown in blue cage) around N-succinylnorvaline and E92 region of X. campestris AOTCase mutants. (E) Contours of the electron density map (2F_o − F_c) (1.0 σ shown in blue cage) around N-acetylnorvaline and P90 region of B. fragilis SOTCase mutant. The final refined positions of the ligands and residues are represented as colored sticks. The residue indicated by * is from the adjacent subunit. The electron density clearly indicates the binding of substrate analogs and the expected amino acid substitutions. (F) N-succinylnorvaline binding site of X. campestris E92A mutant. SNOR is shown as thick ball-and-stick models, while the surrounding residues are shown as thin ball-and-stick models. Two water molecules, Wat7 and Wat31, form hydrogen-bonding bridges to link the ligand to protein residues. These water-mediated hydrogen-bonding networks can only be found in X. campestris AOTCase and its mutants, but not in B. fragilis SOTCase and mutants. The residues, N185 and K302 (X. campestris AOTCase numbering) shown in red, are proposed as the residues that distinguish SOTCase from AOTCase in addition to the key residue of E92.

Figure 2.

Sequence alignments of the regions containing E92, N185, and K302 (shown in bold and indicated by a triangle) to define the substrate specificity of AOTCase and SOTCase. The sequences are from bacteria, Xanthomonas campestris, Xanthomonas oryzae, Xanthomonas axonopodis, Xylella fastidiosa, Parvularcula bermudensis, Maricaulis maris, Oceanicaulis alexandrii, Salinibacter ruber, Psychroflexus torquis, Tenacibaculum sp. Med152, Leeuwenhoekiella blandensis MED217, Polaribacter irgensii 23-P, Flavobacterium johnsoniae UW101, Prevotella ruminicola, Bacteroides thetaiotaomicron, Bacteroides fragilis, Flavobacteriales bacterium BBFL7, Robiginitalea biformata, Tanneralla forsythensis, Myxococcus xanthus, Cellulophaga sp. MED134, Cytophaga hutchinsonii, and Candidatus Sulcia Muelleri.

Figure 3.

A ribbon diagram of the knotted region of X. campestris AOTCase (A) (residues 170–275) and B. fragilis SOTCase (B) (residues 166–255) with bound ligands shown in colored sticks. Colors change continuously from blue (first residue in the region) to red (last residue in the region). The proline-rich loop (from dark blue to light blue), in similar conformation for both AOTCase and SOTCase, appears to be critical for the knot formation. The 240's loop (from brown to red) was threaded through the proline-rich loop to form a trefoil knot.

Figure 4.

Comparison of the canonical and two proposed linear arginine biosynthetic pathways. The canonical pathway is shown in thick arrows.

Figure 5.

A schematic diagram representing the classification of four known transcarbamylases, ATCase, OTCase, AOTCase, and SOTCase, based on structure-derived amino acid signature motifs.