Abstract
CYTH proteins make up a large superfamily that is conserved in all three domains of life. These enzymes have a triphosphate tunnel metalloenzyme (TTM) fold, which typically results in phosphatase functions, e.g., RNA triphosphatase, inorganic polyphosphatase, or thiamine triphosphatase. Some CYTH orthologs cyclize nucleotide triphosphates to 3′,5′-cyclic nucleotides. So far, archaeal CYTH proteins have been annotated as adenylyl cyclases, although experimental evidence to support these annotations is lacking. To address this gap, we characterized a CYTH ortholog, SaTTM, from the crenarchaeote Sulfolobus acidocaldarius. Our in silico studies derived ten major subclasses within the CYTH family implying a close relationship between these archaeal CYTH enzymes and class IV adenylyl cyclases. However, initial biochemical characterization reveals inability of SaTTM to produce any cyclic nucleotides. Instead, our structural and functional analyses show a classical TTM behavior, i.e., triphosphatase activity, where pyrophosphate causes product inhibition. The Ca2+-inhibited Michaelis complex indicates a two-metal-ion reaction mechanism analogous to other TTMs. Cocrystal structures of SaTTM further reveal conformational dynamics in SaTTM that suggest feedback inhibition in TTMs due to tunnel closure in the product state. These structural insights combined with further sequence similarity network–based in silico analyses provide a firm molecular basis for distinguishing CYTH orthologs with phosphatase activities from class IV adenylyl cyclases.
Highlights
Bacterial CyaB-like class IV adenylyl cyclases (AC-IVs) and mammalian thiamine triphosphatases (ThTPases) are founding members of the CyaB-thiamine triphosphatase (CYTH)-like domain superfamily [1]
Archaeal CYTH proteins are functionally diverged from CyaBlike class IV adenylyl cyclases To date, the CYTH-like domain superfamily (IPR033469) comprises 41,000 enzymes from all three domains of life divided into two subfamilies, the smaller mRNA triphosphatase Cet1-like(IPR004206) and the bigger subfamily of CYTH domain proteins (IPR023577) including adenylyl cyclases, ThTPases, and inorganic tri- or polyphosphatases
We aimed to obtain a detailed understanding of the CYTH family by combining biochemical analysis combined with structural snapshots of the catalytic mechanism, as the functional annotations of CYTH enzymes are still vague and misleading among tunnel metalloenzyme (TTM) and adenylyl cyclases
Summary
Bacterial CyaB-like class IV adenylyl cyclases (AC-IVs) and mammalian thiamine triphosphatases (ThTPases) are founding members of the CYTH-like domain superfamily [1]. For triphosphatases and ThTPases, the organic moiety points toward a C-terminal helix, termed plug helix [3, 6] Such enzymes catalyze the hydrolysis of the triphosphate by a two-metal ion mechanism using metal one to establish the catalytically competent binding of the triphosphate moiety and metal two to position and activate a water for nucleophilic attack onto the γ-phosphate moiety [3]. Our comparative structural and bioinformatic analysis of CYTH allowed us to identify molecular determinants, which clearly discriminate the adenylyl cyclase from the different phosphatase activities This provides an improved rationale for correct functional annotation within the broad CYTH domain family
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