S‐Adenosyl‐L‐Methionine Hydrolase (Adenosine‐Forming), a Conserved Bacterial and Archeal Protein Related to SAM‐Dependent Halogenases

Abstract

Keywords DUF62; halogenation; hydrolase; S-adenosyl-L-methionine; SalinisporaS-Adenosyl-L-methionine (SAM) is a ubiquitous molecule that participates in various biochemical reactions, second only to ATP as the most frequently used enzyme substrate. [1] The recently described SAM-dependent halogenases involved in the biosynthesis of secondary metabolites in actinomycetes represent a new family of SAM-binding proteins [2,3] that catalyze the nucleophilic displacement of L-methionine (L-met) from SAM with halides to form halogenated 5′-deoxyadenosine (5′-XDA). [4,5] These enzymes belong to a family of over 100 archaeal and bacterial proteins with no assigned function (pfam 01887, DUF62). Here we report that a DUF62 member from the recently sequenced marine bacterium Salinispora arenicola CNS-205 (SaDUF62, Sare_1364, genome accession number NC_ 009953) has no significant halogenase, but instead SAM hydrolase (adenosine-forming) activity in vitro.SAM is biosynthesized from ATP and L-met by SAM synthetase MetK. Besides its well-known and essential role as a methyl donor to nucleic acids and proteins, [6] the electrophilic character of the carbons surrounding the sulfonium group of SAM makes it also a source of methylene, amino, ribosyl, and aminoalkyl groups, as well as 5′-deoxyadenosyl radicals. [1] Furthermore, SAM acts as a molecular effector in the riboswitch-mediated feedback regulation of metK and methionine biosynthesis genes in bacteria [7,8] and plants. [9] Yet, the regulatory role of SAM is not limited to the met operon. SAM levels have been shown to influence morphological differentiation and secondary-metabolite biosynthesis in the soil bacteria Streptomyces-high levels of SAM cause antibiotic overproduction and inhibit sporulation-at least in part by promoting transcription of regulatory genes. [10][11][12] Correspondence to: Alessandra S. Eustáquio. Note Added in ProofDuring revision of this manuscript, a similar study by Deng et al. [29] describing the in vitro characterization of PhDUF62 was published. The authors show that while PhDUF62 is unable to catalyze fluorination or chlorination reactions, it does convert SAM to adenosine in vitro. Assay of the enzyme in H 218 O results in [5′-18 O]adenosine without labeling of the protein; this indicates that a mechanism of the type depicted in Figure 2 is operating rather than a second possibility through an enzyme-bound intermediate. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptWe recently reported that biosynthesis of the marine bacterial natural product salinosporamide A from Salinispora tropica involves a SAM-dependent chlorinase [5] similar to fluorinase from the fluoroacetate-producer Streptomyces cattleya. [4] These two enzymes represent yet another example of the versatility of SAM-binding proteins, [2,3] and are the only members characterized from a family of over 100 bacterial and archaeal homologues available in GenBank and assigned as "protein of unknown function, DUF62, pfam 01887". Given their wide distribution in the b...

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S -adenosyl- l -methionine

“…We recently showed that a DUF62 ortholog from Salinispora arenicola (86% identity to Strop_1405) has SAM hydrolase but no halogenase activity in vitro (33), which was independently reported with an archaeal DUF62 (34). Together with in silico analysis, these data suggest divergent evolution of SAM-dependent halogenases from SAM hydrolases (33) and clarify the salL mutant phenotype.…”

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

“…Protein sequence alignments show that Gly131 (SalL numbering) is conserved in most cases, while Tyr70 is replaced by nonpolar, aliphatic residues including Val, Gly and Ile. 306–308 On the basis of structure–function studies of FlA and SalL, these DUF62 proteins would most likely not act as nucleophilic halogenases due to the predicted larger size of the halide binding pocket and likely presence of water in the active site. In fact, two DUF62 proteins have been biochemically analyzed, i.e.…”

“…Only iodide was accepted by SARE1364 but with K m for iodide (20 mM, which is 40,000-fold higher than the concentration of iodide in seawater) and k cat (0.5 h −1 ) that do not appear biologically relevant. 306 Moreover, DUF62 proteins contain a strictly conserved Asp-Arg-His triad that is absent in fluorinase and chlorinase and is proposed to be involved in water activation for SAM hydrolysis (Figure 31). 306–308 It remains to be shown if this SAM hydrolase activity measured in vitro is physiologically relevant.…”

Salinosporamid‐Naturstoffe: potente Inhibitoren des 20S‐Proteasoms als vielversprechende Krebs‐Chemotherapeutika

“…Der Einbau von Fluorid ist allerdings wegen subtiler Umlagerungen in der Halogenbindungstasche, wie sie bei hochaufgelösten Kristallstrukturen von SalL und Mutanten des aktiven Zentrums beobachtet wurden, mit SalL nicht möglich. Zusammen mit der Fluorinase FlA [68] und den kürzlich entdeckten SAM-Hydroxid-Adenosyltransferasen (duf-62) [70][71][72] bildet SalL eine neue Klasse SAM metabolisierender Enzyme. [73] Die Bildung von 5'-Chlor-5'-desoxyadenosin (49, 5-ClDA) initiiert eine komplexe Biosyntheseroute zu 47, wobei alle dafür entscheidenden Gene im salCluster zu finden sind (Schema 5 a, blau eingefärbt), wie durch Gen-Inaktivierung und chemische Komplementierung aufgezeigt wurde.…”