Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase.


Abstract

Predictions of tertiary structures of proteins from their amino acid sequences are facilitated greatly when the structures of homologous proteins are known. On this basis, structural features of Escherichia coli ornithine transcarbamoylase (OTCase) were investigated by site-directed mutagenesis experiments based on the known tertiary structure of the catalytic (c) chain of E. coli aspartate transcarbamoylase (ATCase). In ATCase, each c chain is composed of two globular domains connected by two interdomain helices, one of which is near the C-terminus and is critical for the in vivo folding of the chains and their assembly into trimers. Each active site is located at the interface between two chains and requires the participation of residues from each of the adjacent chains. OTCase, a trimeric enzyme, has been proposed to be similar in structure to the ATCase trimer on the basis of sequence identity (32%), the nature of the reaction catalyzed by the enzyme, and secondary structure predictions. As shown here, analysis of OTCase and ATCase sequences revealed extensive evolutionary conservation in portions corresponding to the ATCase active site and the C-terminal helix. Truncations and substitutions within the predicted C-terminal helix of OTCase had effects on activity and thermal stability strikingly similar to those caused by analogous alterations in ATCase. Similarly, substitutions at either of two conserved residues, Ser 55 and Lys 86, in the proposed active site of OTCase had deleterious effects parallel to those caused by the analogous ATCase substitutions. Hybrid trimers comprised of chains from both these relatively inactive OTCase mutants exhibited dramatically increased activity, as predicted for shared active sites located at the chain interfaces. These results strongly support the hypothesis that the tertiary and quaternary structures of the two enzymes are similar. Study holds ProTherm entries: 9284, 9285, 9286, 9287, 9288 Extra Details: additive : EDTA(0.2 mM),beta mercaptoethanol(5 mM) was added in the experiment C-terminal helix; homology; shared active sites;,structure prediction

Submission Details

ID: XZPs4spU3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Murata LB;Schachman HK,Protein Sci. (1996) Structural similarity between ornithine and aspartate transcarbamoylases of Escherichia coli: characterization of the active site and evidence for an interdomain carboxy-terminal helix in ornithine transcarbamoylase. PMID:8845761
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1DUV 2000-07-04 1.7 CRYSTAL STRUCTURE OF E. COLI ORNITHINE TRANSCARBAMOYLASE COMPLEXED WITH NDELTA-L-ORNITHINE-DIAMINOPHOSPHINYL-N-SULPHONIC ACID (PSORN)
2OTC 1998-06-17 2.8 ORNITHINE TRANSCARBAMOYLASE COMPLEXED WITH N-(PHOSPHONACETYL)-L-ORNITHINE
1AKM 1998-05-27 2.8 ORNITHINE TRANSCARBAMYLASE FROM ESCHERICHIA COLI

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.0 Ornithine carbamoyltransferase subunit I Q8FAD6 OTC_ECOL6
98.5 Ornithine carbamoyltransferase subunit I Q328S8 OTC_SHIDS
98.5 Ornithine carbamoyltransferase subunit I B7MLQ9 OTC_ECO45
98.5 Ornithine carbamoyltransferase subunit I A7ZVD3 OTC_ECO24
99.1 Ornithine carbamoyltransferase subunit I Q0SXJ4 OTC_SHIF8
99.1 Ornithine carbamoyltransferase subunit I Q8XCB8 OTC_ECO57
99.1 Ornithine carbamoyltransferase subunit I Q3YU98 OTC_SHISS
99.1 Ornithine carbamoyltransferase subunit I Q31TJ4 OTC_SHIBS
99.4 Ornithine carbamoyltransferase subunit I Q83IM2 OTC_SHIFL
99.4 Ornithine carbamoyltransferase subunit I B7NGI6 OTC_ECOLU
99.4 Ornithine carbamoyltransferase subunit I B1ISV4 OTC_ECOLC
99.4 Ornithine carbamoyltransferase subunit I A8A810 OTC_ECOHS
99.4 Ornithine carbamoyltransferase subunit I B7M9L3 OTC_ECO8A
99.4 Ornithine carbamoyltransferase subunit I B7LCW4 OTC_ECO55
100.0 Ornithine carbamoyltransferase subunit I P04391 OTC1_ECOLI