Structural Determinants for Substrate Selectivity in Guanine Deaminase Enzymes of the Amidohydrolase Superfamily

Roger Shek, Tylene Hilaire, Jasper Sim, Jarrod B. French

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Guanine deaminase is a metabolic enzyme, found in all forms of life, which catalyzes the conversion of guanine to xanthine. Despite the availability of several crystal structures, the molecular determinants of substrate orientation and mechanism remain to be elucidated for the amidohydrolase family of guanine deaminase enzymes. Here, we report the crystal structures of Escherichia coli and Saccharomyces cerevisiae guanine deaminase enzymes (EcGuaD and Gud1, respectively), both members of the amidohydrolase superfamily. EcGuaD and Gud1 retain the overall TIM barrel tertiary structure conserved among amidohydrolase enzymes. Both proteins also possess a single zinc cation with trigonal bipyrimidal coordination geometry within their active sites. We also determined a liganded structure of Gud1 bound to the product, xanthine. Analysis of this structure, along with kinetic data of native and site-directed mutants of EcGuaD, identifies several key residues that are responsible for substrate recognition and catalysis. In addition, after a small library of compounds had been screened, two guanine derivatives, 8-azaguanine and 1-methylguanine, were identified as EcGuaD substrates. Interestingly, both EcGuaD and Gud1 also exhibit secondary ammeline deaminase activity. Overall, this work details key structural features of substrate recognition and catalysis of the amidohydrolase family of guanine deaminase enzymes in support of our long-term goal to engineer these enzymes with altered activity and substrate specificity.

Original languageEnglish (US)
Pages (from-to)3280-3292
Number of pages13
JournalBiochemistry
Volume58
Issue number30
DOIs
StatePublished - Jul 30 2019
Externally publishedYes

Bibliographical note

Funding Information:
This work was funded in part by the National Institute of General Medical Sciences of the National Institutes of Health (Grant R35GM124898 to J.B.F.).

Funding Information:
The authors thank Anne Ostermeyer-Fay for help with cloning, Dr. Ed Luk for the kind donation of yeast cDNA, and the staff at NE-CAT and AMX for support and direction with data collection. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences of the National Institutes of Health (P41 GM103403). The Pilatus 6M detector on beamline 24-ID-C is funded by a NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357, and the AMX and FMX beamlines of National Synchrotron Light Source II, a DOE Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract DE-SC0012704.

Publisher Copyright:
© 2019 American Chemical Society.

Fingerprint Dive into the research topics of 'Structural Determinants for Substrate Selectivity in Guanine Deaminase Enzymes of the Amidohydrolase Superfamily'. Together they form a unique fingerprint.

Cite this