Crystal structure of an ASCH protein from Zymomonas mobilis and its ribonuclease activity specific for single-stranded RNA

Abstract

Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central β-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the β-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5′-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.

Modified base-binding EVE and DCD domains: striking diversity of genomic contexts in prokaryotes and predicted involvement in a variety of cellular processes

“…Sequence and structure analyses have shown that the EVE domain is a member of the PUA ( p seudo u ridine synthase and a rchaeosine transglycosylase)/ASCH ( ASC -1 h omology) superfamily, a widely disseminated and apparently ancient assemblage of nucleic acid-binding domains [ 8 – 13 ]. These domains are generally associated with the translation apparatus, often fused to RNA modification enzymes, and bind RNA themselves [ 11 – 14 ]. Some ASCH domains have also been predicted to bind modified bases [ 15 ].…”

YqfB protein from Escherichia coli: an atypical amidohydrolase active towards N4-acylcytosine derivatives

“…Recently, the crystal structure of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH) has been determined revealing a ribonuclease activity. The detailed mechanism of RNA hydrolysis is yet to be elucidated, but it has been confirmed that three residues (Tyr47, Lys53, and Ser128) located in a cleft contribute to nucleic acid-binding and RNA cleavage 25 . However, the biological role Media and growth conditions.…”

A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

“…The amino acid sequence analysis using SMART [46] revealed that D8_RL contains the human activating signal cointegrator homology (ASCH) [47] and growth-arrest-specific protein 2 (GAS2) domains [48]. ASCH domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them [47], except for an ASCH domain-containing ribonuclease from Zymomonas mobilis [49] and a recently characterized unique ASCH domain-containing amidohydrolase YqfB from Escherichia coli, which is active towards N 4 -acylcytidines [50]. The comparison of YqfB and D8_RL amino acid sequences showed merely 17 identical residues between these two proteins, and only 2 of the 4 YqfB catalytic amino acids were among them, although the aspartic acid residue in position 72 of the D8_RL and the catalytic glutamic acid residue in position 74 of the YqfB enzyme can also be considered conservative ones (Figure 3 and Figure S2).…”