Open AccessCloning, purification, crystallization and preliminary X‐ray crystallographic analysis of the biosynthetic N ‐acetylornithine aminotransferases from Salmonella typhimurium and Escherichia coli
Author(s) -
Rajaram V.,
Prasad K.,
Ramachandra N.,
Bharath S. R.,
Savithri H. S.,
Ratna Prasuna P.,
Murthy M. R. N.
Publication year - 2006
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309106033884
Subject(s) - escherichia coli , arginine , pyridoxal , biochemistry , biosynthesis , dimer , enzyme , crystallization , chemistry , ornithine , salmonella , biology , stereochemistry , amino acid , bacteria , gene , organic chemistry , genetics
Acetylornithine aminotransferase (AcOAT) is a type I pyridoxal 5′‐phosphate‐dependent enzyme catalyzing the conversion of N ‐acetylglutamic semialdehyde to N ‐acetylornithine in the presence of α‐ketoglutarate, a step involved in arginine metabolism. In Escherichia coli , the biosynthetic AcOAT also catalyzes the conversion of N ‐succinyl‐ l ‐2‐amino‐6‐oxopimelate to N ‐succinyl‐ l , l ‐diaminopimelate, one of the steps in lysine biosynthesis. It is closely related to ornithine aminotransferase. AcOAT was cloned from Salmonella typhimurium and E. coli , overexpressed in E. coli and purified using Ni–NTA affinity column chromatography. The enzymes crystallized in the presence of gabaculine. Crystals of E. coli AcOAT (eAcOAT) only diffracted X‐rays to 3.5 Å and were twinned. The crystals of S. typhimurium AcOAT (sAcOAT) diffracted to 1.9 Å and had a dimer in the asymmetric unit. The structure of sAcOAT was solved by the molecular‐replacement method.