Abstract
S-adenosyl-L-homocysteine (SAH) hydrolases (SAHases) are involved in the regulation of methylation reactions in many organisms and are thus crucial for numerous cellular functions. Consequently, their dysregulation is associated with severe health problems. The SAHase-catalyzed reaction is reversible and both directions depend on the redox activity of nicotinamide adenine dinucleotide (NAD+) as a cofactor. Therefore, nicotinamide cofactor biomimetics (NCB) are a promising tool to modulate SAHase activity. In the present in vitro study, we investigated 10 synthetic truncated NAD+ analogs against a SAHase from the root-nodulating bacterium Bradyrhizobium elkanii. Among this set of analogs, one was identified to inhibit the SAHase in both directions. Isothermal titration calorimetry (ITC) and crystallography experiments suggest that the inhibitory effect is not mediated by a direct interaction with the protein. Neither the apo-enzyme (i.e., deprived of the natural cofactor), nor the holo-enzyme (i.e., in the NAD+-bound state) were found to bind the inhibitor. Yet, enzyme kinetics point to a non-competitive inhibition mechanism, where the inhibitor acts on both, the enzyme and enzyme-SAH complex. Based on our experimental results, we hypothesize that the NCB inhibits the enzyme via oxidation of the enzyme-bound NADH, which may be accessible through an open molecular gate, leaving the enzyme stalled in a configuration with oxidized cofactor, where the reaction intermediate can be neither converted nor released. Since the reaction mechanism of SAHase is quite uncommon, this kind of inhibition could be a viable pharmacological route, with a low risk of off-target effects. The NCB presented in this work could be used as a template for the development of more potent SAHase inhibitors.
Highlights
Synthetic cofactor analogs proved their potential to replace the natural nicotinamide adenine dinucleotide cofactor NAD(P)H in certain oxidoreductases (Paul et al, 2013, 2014; Paul and Hollmann, 2016)
Recent studies have shown that the reduced form of certain nicotinamide cofactor biomimetics (NCB) can reconstitute activity in several oxidoreductases in the absence of the natural nicotinamide adenine dinucleotide cofactor (Paul et al, 2013, 2014; Paul and Hollmann, 2016; Nowak et al, 2017b)
A screen of the NCBs with holo-S-adenosylL-homocysteine hydrolase (SAHase) revealed that NCB 10 led to significantly lower substrate conversion (∼75% inhibition with 2 mM of 10), whereas the other analogs did not affect the enzymatic activity
Summary
Synthetic cofactor analogs proved their potential to replace the natural nicotinamide adenine dinucleotide (phosphate) cofactor NAD(P)H in certain oxidoreductases (Paul et al, 2013, 2014; Paul and Hollmann, 2016). Whether the analogs were used to modulate the electron transfer process (Paul et al, 2015) or the kinetics of the enzymatic reaction (Knaus et al, 2016), Nicotinamide Cofactor Biomimetic Inhibits SAHase their activity is of utmost importance (Li et al, 2009). We present the screening of several synthetic truncated NCBs and their effect on the activity of the SAHase homolog from Bradyrhizobium elkanii as a model enzyme. Since one of the NCBs turned out to be an inhibitor, a thorough characterization of the interactions with the enzyme has been carried out to elucidate the inhibition mechanism
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