Urea producing bimetallic arginases are essential for the synthesis of polyamine, DNA, and RNA. Despite conservation of the signature motifs in all arginases, a nonconserved 153ESEEKAWQKLCSL165 motif is found in the Helicobacter pylori enzyme, whose role is yet unknown. Using site-directed mutagenesis, kinetic assays, metal analyses, circular dichroism, heat-induced denaturation, molecular dynamics simulations and truncation studies, we report here the significance of this motif in catalytic function, metal retention, structural integrity, and stability of the protein. The enzyme did not exhibit detectable activity upon deletion of the motif as well as on individual mutation of Glu155 and Trp159 while Cys163Ala displayed significant decrease in the activity. Trp159Ala and Glu155Ala show severe loss of thermostability (14–17°) by a decrease in the α-helical structure. The role of Trp159 in stabilization of the structure with the surrounding aromatic residues is confirmed when Trp159Phe restored the structure and stability substantially compared to Trp159Ala. The simulation studies support the above results and show that the motif, which was previously solvent exposed, displays a loop-cum-small helix structure (Lys161–Cys163) and is located near the active-site through a novel Trp159–Asp126 interaction. This is consistent with the mutational analyses, where Trp159 and Asp126 are individually critical for retaining a bimetallic center and thereby for function. Furthermore, Cys163 of the helix is primarily important for dimerization, which is crucial for stimulation of the activity. Thus, these findings not only provide insights into the role of this motif but also offer a possibility to engineer it in human arginases for therapeutics against a number of carcinomas.

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Natural antisense long noncoding RNAs (lncNATs) are involved in the regulation of gene expression in plants, modulating different relevant developmental processes and responses to various stimuli. We identified and characterized two lncNATs (NAT1UGT73C6 and NAT2UGT73C6, collectively NATsUGT73C6) in Arabidopsis thaliana that are transcribed from a gene overlapping UGT73C6, a member of the UGT73C subfamily of genes encoding UDP-glycosyltransferases (UGTs). Expression of both NATsUGT73C6 is developmentally controlled and occurs independently of the transcription of UGT73C6 in cis. Downregulation of NATsUGT73C6 levels through artificial microRNAs results in a reduction of the rosette area, while constitutive overexpression of NAT1UGT73C6 or NAT2UGT73C6 leads to the opposite phenotype, an increase in rosette size. This activity of NATsUGT73C6 relies on its RNA sequence, and, although modulation of UGT73C6 in cis cannot be excluded, the observed phenotypes are not a consequence of the regulation of UGT73C6 in trans. The NATsUGT73C6 levels were shown to affect cell proliferation and thus individual leaf size. Consistent with this concept, our data suggest that the NATsUGT73C6 modulate the expression levels of key transcription factors involved in regulating leaf growth by modulating cell proliferation. These findings thus reveal an additional regulatory layer on the process of leaf growth.

SUMMARY Natural antisense long non‐coding RNAs (lncNATs) are involved in the regulation of gene expression in plants, modulating different relevant developmental processes and responses to various stimuli. We have identified and characterized two lncNATs (NAT1UGT73C6 and NAT2UGT73C6, collectively NATsUGT73C6) from Arabidopsis thaliana that are transcribed from a gene fully overlapping UGT73C6, a member of the UGT73C subfamily of genes encoding UDP‐glycosyltransferases (UGTs). Expression of both NATsUGT73C6 is developmentally controlled and occurs independently of the transcription of UGT73C6 in cis. Downregulation of NATsUGT73C6 levels through artificial microRNAs results in a reduction of the rosette area, while constitutive overexpression of NAT1UGT73C6 or NAT2UGT73C6 leads to the opposite phenotype, an increase in rosette size. This activity of NATsUGT73C6 relies on its RNA sequence and, although modulation of UGT73C6 in cis cannot be excluded, the observed phenotypes are not a consequence of the regulation of UGT73C6 in trans. The NATsUGT73C6 levels were shown to affect cell proliferation and thus individual leaf size. Consistent with this concept, our data suggest that the NATsUGT73C6 influence the expression levels of key transcription factors involved in regulating leaf growth by modulating cell proliferation. These findings thus reveal an additional regulatory layer on the process of leaf growth. In this work, we characterized at the molecular level two long non‐coding RNAs (NATsUGT73C6) that are transcribed in the opposite direction to UGT73C6, a gene encoding a glucosyltransferase involved in brassinosteroid homeostasis in A. thaliana. Our results indicate that NATsUGT73C6 expression influences leaf growth by acting in trans and by modulating the levels of transcription factors that are involved in the regulation of cell proliferation.