Crystal structure and biochemical properties of putrescine carbamoyltransferase from Enterococcus faecalis: Assembly, active site, and allosteric regulation

Abstract

Putrescine carbamoyltransferase (PTCase) catalyzes the conversion of carbamoylputrescine to putrescine and carbamoyl phosphate (CP), a substrate of carbamate kinase (CK). The crystal structure of PTCase has been determined and refined at 3.2 Å resolution. The trimeric molecular structure of PTCase is similar to other carbamoyltransferases, including the catalytic subunit of aspartate carbamoyltransferase (ATCase) and ornithine carbamoyltransferase (OTCase). However, in contrast to other trimeric carbamoyltransferases, PTCase binds both CP and putrescine with Hill coefficients at saturating concentrations of the other substrate of 1.53 ± 0.03 and 1.80 ± 0.06, respectively. PTCase also has a unique structural feature: a long C-terminal helix that interacts with the adjacent subunit to enhance intersubunit interactions in the molecular trimer. The C-terminal helix appears to be essential for both formation of the functional trimer and catalytic activity, since truncated PTCase without the C-terminal helix aggregates and has only 3% of native catalytic activity. The active sites of PTCase and OTCase are similar, with the exception of the 240’s loop. efPTCase lacks the proline-rich sequence found in knotted carbamoyltransferases and is unknotted. A Blast search of all available genomes indicates that 35 bacteria, most of which are Gram-positive, have an agcB gene encoding PTCase located near the genes that encode agmatine deiminase and carbamate kinase, consistent with the catabolic role of PTCase in the agmatine degradation pathway. The C-terminal helix identified in efPTCase is found in all other PTCases identified, suggesting that it is the signature feature of the PTCase family of enzymes.

From Genome to Structure and Back Again: A Family Portrait of the Transcarbamylases

“…Putrescine transcarbamylase— Five structures of PTCase from E. faecalis with and without ligands have been determined that provide significant insight into its structure, function and mechanism [ 29 , 30 ].…”

“…PTCase’s special feature is an extra long helix at its C-terminal end, which extends to cover helix 1 of the adjacent subunit ( Figure 4 ). This feature appears to be important in stabilizing the catalytic trimer and preventing formation of a larger oligomer [ 29 , 30 ].…”

“…In PTCase, structure determination clearly revealed that D227 and Y233 from the equivalent 230’s loop and Q164, which hydrogen bonds to the amino group, directly shape the putrescine binding site with participation of E236 and H83 * from the adjacent subunit ( Figure 6 C) [ 29 ]. However, residues Y233, E236 and H83 * are not conserved in other PTCase sequences [ 30 ]. How different residues shape the putrescine binding site and whether other hypothetical PTCases with sequence variations are true PTCases remains to be established.…”

“…This is expected for AOTCase and SOTCase since both of them play an anabolic role in the arginine biosynthetic pathway [ 21 , 24 ]. Even though PTCase and YTCase were proposed to play a catabolic role, both function as homotrimers [ 29 , 30 , 32 ]. Both PTCase and YTCase have one interesting common structural feature; the first N-terminal helix (the equivalent helix is the second helix in the YTCase structure because of the presence of an extra N-terminal helix) is covered by other helices.…”

“…The assignment of PTCase of Enterococcus faecalis was recently confirmed enzymatically and structurally [ 28 , 29 ]. The liganded and unliganded structures indicate that the active subunit is trimeric, similar to anabolic OTCase, AOTCase and SOTCase [ 29 , 30 ].…”