Luciferase-Based Assay for Adenosine: Application to S-Adenosyl-l-homocysteine Hydrolase

Abstract

S-adenosyl-L-homocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosyl-L-homocysteine (SAH) to adenosine (ADO) and L-homocysteine (Hcy), promoting methyltransferases activity by relief of SAH inhibition. SAH catabolism is linked to S-adenosylmethionine metabolism and the development of SAHH inhibitors is of interest for new therapeutics with anti-cancer or cholesterol-lowering effects. We have developed a continuous enzymatic assay for adenosine that facilitates high-throughput analysis of SAHH. This luciferase-based assay is 4000-fold more sensitive than former detection methods and is well suited for continuous monitoring of ADO formation in a 96 well plate format. The high-affinity adenosine kinase from Anopheles gambiae (AgAK) efficiently converts adenosine to AMP in the presence of GTP. AMP is converted to ATP and coupled to firefly luciferase. With this procedure, kinetic parameters (Km, kcat) for SAHH were obtained, in good agreement with literature values. Assay characteristics include sustained light output combined with ultra-sensitive detection (10−7 unit SAHH). The assay is documented with the characterization of slow-onset inhibition for inhibitors of the hydrolase. Application of this assay may facilitate the development of SAHH inhibitors and provide an ultrasensitive detection for the formation of adenosine from other biological reactions.

A rapid enzymatic assay for high‐throughput screening of adenosine‐producing strains

“…In comparison with the traditional qualitative and quantitative detection of adenosine using chromatography methods such as HPLC, which are expensive, laborious and low-throughput (10 2 –10 4 colonies per week), the reported assay is high-throughput, quick, sensitive and highly adaptable systems (10 6 –10 8 colonies per week) (Strege, 1999 ; Pham-Tuan et al ., 2003 ). The methods developed for determination of adenosine in serum, urine or tissue (Kloor et al ., 2000 ; Burgos et al ., 2012 ; Helenius et al ., 2012 ; Li et al ., 2012 ; Wang et al ., 2012 ; Zhang et al ., 2013 ) have nice detection limit and linear relationship. However, the detection range is too narrow (see Table S1 ) and the test cost is too high for them to be used in detecting the adenosine in fermentation broth in large numbers of samples.…”

Involvement of Sulfur in the Biosynthesis of Essential Metabolites in Pathogenic Fungi of Animals, Particularly Aspergillus spp.: Molecular and Therapeutic Implications

“…Thus, a number of unforeseen interactions are emerging between primary and so-called secondary metabolism, which may result in new anti-fungal drug target identification. Since SAM is essential for many cellular transmethylation reactions (e.g., epigenetic regulation) (Strauss and Reyes-Dominguez, 2011), and SAH may be an inhibitor of cellular methyltransferases (Burgos et al, 2012), there may be significant antifungal drug target potential associated with the overproduction of BmGT or inhibition of GliK activity-both of which may cause SAM dissipation. In addition, gliotoxin, or specific reactions required for its biosynthesis, have also been shown to influence the formation of other sulfur-containing metabolites, like EGT (Gallagher et al, 2012;Sheridan et al, 2016), and finally, the activity of gliotoxin against fungi is revealing even further interactions within biological systems-most recently involving zinc homeostasis (Vicentefranqueira et al, 2018).…”

Proteomic analysis of Aspergillus fumigatus – clinical implications

“…Thus, proteomics has revealed a wealth of previously occluded interactions between primary and secondary metabolism, many of which have potential for exploitation in the clinical setting as anti-fungal drug targets. Indeed, given the essential nature of SAM for a plethora of cellular transmethylation reactions including, but not limited to, epigenetic regulation [85], and the potential inhibitory action of SAH on cellular methyltransferases [86], the metabolically catastrophic potential of dysregulating GliT-mediated control of gliotoxin/BmGT biosynthesis is only just becoming apparent. Further systems impacts of interfering with gliotoxin biosynthesis are only just emerging and, surprisingly, it appears that the biosynthesis of apparently unrelated natural products, like the antioxidant ergothioneine, is influenced either by gliotoxin [87,88] (and unpublished data), or specific reactions within its biosynthetic pathway [72].…”