Altered specificity of Hint-W123Q supports a role for Hint inhibition by ASW in avian sex determination

Abstract

Hint is a universally conserved, dimeric AMP-lysine hydrolase encoded on the avian Z chromosome. Tandemly repeated on the female-specific W chromosome, Asw encodes a potentially sex-determining, dominant-negative Hint dimerization partner whose substrate-interacting residues were specifically altered in evolution. To test the hypothesis that Gln127 of Asw is responsible for depression and/or alteration of Hint enzyme activity, a corresponding mutant was created in the chicken Hint homodimer, and a novel substrate was developed that links reversal of AMP-lysine modification to aminomethylcoumarin release. Strikingly, the Hint-W123Q substitution reduced k(cat)/K(m) for AMP-lysine hydrolysis 17-fold, while it increased specificity for AMP-para-nitroaniline hydrolysis by 160-fold. The resulting 2,700-fold switch in enzyme specificity suggests that Gln127 could be the dominant component of Asw dominant negativity in avian feminization.

Genetic evidence against a role for W-linked histidine triad nucleotide binding protein (HINTW) in avian sex determination

“…However, the predicted HINTW protein lacks the key catalytic motif common to all other HINT enzymes. Several in vitro biochemical studies have shown that over-expression of the predicted HINTW protein blocks the function of the Z-linked homologue, HINTZ (Moriyama et al, 2006;Pace and Brenner, 2003;Parks et al, 2004). HINTZ encodes a bona fide HINT protein that has adenosine monophosphoramidase activity, but it can heterodimerise with HINTW in vitro, causing a dramatic reduction in its function.…”

31P NMR and Genetic Analysis Establish hinT as the Only Escherchia coli Purine Nucleoside Phosphoramidase and as Essential for Growth under High Salt Conditions

“…Extensive analysis of their metabolism by cell extracts and whole cells has provided supporting evidence that the hydrolysis of the P-N bond necessary for subsequent phosphorylations is enzyme-mediated. Recently, yeast Hnt1, rabbit Hint1, and chicken Hint have been shown to be AMP-lysine hydrolases, suggesting that this class of enzymes may be responsible for nucleoside phosphoramidase activities (3)(4)(5). A test of this hypothesis required development of an assay that could measure total cellular nucleoside monophosphoramidate hydrolase activity and a genetic system to knock out candidate genes.…”

“…The Hint branch, named for similarity with rabbit Hint1, 1 is the most ancient branch, having representatives in all forms of life (1,2). The best biochemical substrates of these enzymes are model compounds containing a adenylylated-lysine residue from which Hint hydrolyzes AMP, leaving unmodified lysine (3)(4)(5). Deletions or point mutations that target the enzymatic activity of Saccharomyces cerevisiae homolog hnt1 result in cells that are temperature-sensitive on galactose media and are hypersensitive to mutations in components of the general transcription factor transcription factor II H (namely Kin28, Ccl1, Tfb3) and to mutations in the cyclindependent kinase-activating kinase Cak1 (3).…”

“…Deletions or point mutations that target the enzymatic activity of Saccharomyces cerevisiae homolog hnt1 result in cells that are temperature-sensitive on galactose media and are hypersensitive to mutations in components of the general transcription factor transcription factor II H (namely Kin28, Ccl1, Tfb3) and to mutations in the cyclindependent kinase-activating kinase Cak1 (3). Birds encode a typical HINT gene on the Z chromosome, such that males have two doses, whereas avian ASW genes, which may function as dominant negative inhibitors of Hint via Gln-127, are repeated 40 times on the female-specific W chromosome (5,6). The hint1 homolog of mouse is a nonessential gene (7), the loss of which predisposes to an increased incidence of tumors (8).…”

Rowley Review

“…It has been suggested that HINTW may function in avian sex determination by inhibiting the (malepromoting) activity of HINTZ (Pace and Brenner 2003). In vitro enzyme data support a dominant negative influence of HINTW on HINTZ enzymatic activity (Parks et al 2004). However, the HINTW gene is apparently absent in ratites, and overexpression in early Chicken embryos does not induce ovarian development, undermining this gene as a candidate female determinant (Smith et al 2009b).…”