Document Detail


The structure of allophanate hydrolase from Granulibacter bethesdensis provides insights into substrate specificity in the amidase signature family.
MedLine Citation:
PMID:  23282241     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Allophanate hydrolase (AH) catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH(3) and CO(2). AH belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cis-Ser-Lys catalytic triad. In this study, the first structures of AH from Granulibacter bethesdensis were determined, with and without the substrate analogue malonate, to 2.2 and 2.8 Å, respectively. The structures confirm the identity of the catalytic triad residues and reveal an altered dimerization interface that is not conserved in the amidase signature family. The structures also provide insights into previously unrecognized substrate specificity determinants in AH. Two residues, Tyr(299) and Arg(307), are within hydrogen bonding distance of a carboxylate moiety of malonate. Both Tyr(299) and Arg(307) were mutated, and the resulting modified enzymes revealed >3 order of magnitude reductions in both catalytic efficiency and substrate stringency. It is proposed that Tyr(299) and Arg(307) serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser(172). The structure further suggests the presence of a unique C-terminal domain in AH. While this domain is conserved, it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Analysis of the AH active site architecture offers new insights into common determinants of catalysis and specificity among divergent members of the amidase signature family.
Authors:
Yi Lin; Martin St Maurice
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.     Date:  2013-01-18
Journal Detail:
Title:  Biochemistry     Volume:  52     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-29     Completed Date:  2013-03-25     Revised Date:  2014-01-30    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  United States    
Other Details:
Languages:  eng     Pagination:  690-700     Citation Subset:  IM    
Data Bank Information
Bank Name/Acc. No.:
PDB/4GYR;  4GYS
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Acetobacteraceae / enzymology*
Allophanate Hydrolase / chemistry*,  genetics
Amino Acid Sequence
Amino Acid Substitution
Apoenzymes / chemistry
Bacterial Proteins / chemistry*,  genetics
Catalytic Domain
Conserved Sequence
Crystallography, X-Ray
Models, Molecular
Molecular Sequence Data
Mutagenesis, Site-Directed
Protein Structure, Quaternary
Substrate Specificity
Thermodynamics
Grant Support
ID/Acronym/Agency:
R15 GM097724/GM/NIGMS NIH HHS; R15GM097724/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Apoenzymes; 0/Bacterial Proteins; EC 3.5.1.54/Allophanate Hydrolase
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Human endogenous retroviruses and cancer prevention: evidence and prospects.
Next Document:  Relationship between intra-abdominal pressure and indocyanine green plasma disappearance rate: hepat...