Crystal structure and biochemical properties of putrescine carbamoyltransferase from Enterococcus faecalis: Assembly, active site, and allosteric regulation.
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. PTCase 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 CK, consistent with the catabolic role of PTCase in the agmatine degradation pathway. Sequence comparisons indicate that the C-terminal helix identified in this PTCase structure will be found in all other PTCases identified, suggesting that it is the signature feature of the PTCase family of enzymes.
Center for Genetic Medicine Research and Department of Integrative Systems Biology, Children's National Medical Center, The George Washington University, Washington, District of Columbia 20010, USA. dshi@cnmcresearch.org
Dashuang
D
Shi
Xiaolin
X
Yu
Gengxiang
G
Zhao
Jeremy
J
Ho
Shennon
S
Lu
Norma M
NM
Allewell
Mendel
M
Tuchman
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D003460:Crystallization; D030541:Databases, Genetic; D013293:Enterococcus faecalis / Q000201:enzymology* / Q000378:metabolism; D006639:Histidine; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase; D011487:Protein Conformation; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D006639:Histidine; D019878:Carboxyl and Carbamoyl Transferases; D009954:Ornithine Carbamoyltransferase; C022487:putrescine carbamoyltransferase