title: The structure of apo ArnA features an unexpected central binding pocket and provides an explanation for enzymatic cooperativity.
abstract: The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD(+)-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.
authors.affiliation: Department of Biochemistry, Biocenter of the University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Utz
authors.initials: U
authors.lastname: Fischer
authors.affiliation: Department of Biochemistry, Biocenter of the University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Hertlein
authors.affiliation: Department of Biochemistry, Biocenter of the University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Clemens
authors.initials: C
authors.lastname: Grimm
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D009243:NAD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D009243:NAD; D014535:Uridine Diphosphate Glucuronic Acid; D000255:Adenosine Triphosphate; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase
keywords: ArnA