Dataset Information

A High-Throughput Method for Directed Evolution of NAD(P)+-Dependent Dehydrogenases for the Reduction of Biomimetic Nicotinamide Analogues

ABSTRACT: Engineering flavin-free NAD(P)+-dependent dehydrogenases to reduce biomimetic nicotinamide analogues (mNAD+s) is of importance for eliminating the need for costly NAD(P)+ in coenzyme regeneration systems. Current redox dye-based screening methods for engineering the mNAD+ specificity of dehydrogenases are frequently encumbered by a background signal from endogenous NAD(P) and intracellular reducing compounds, making the detection of low mNAD+-based activities a limiting factor for directed evolution. Here, we develop a high-throughput screening method, NAD(P)-eliminated solid-phase assay (NESPA), which can reliably identify mNAD+-active mutants of dehydrogenases with a minimal background signal. This method involves (1) heat lysis of colonies to permeabilize the cell membrane, (2) colony transfer onto filter paper, (3) washing to remove endogenous NAD(P) and reducing compounds, (4) enzyme-coupled assay for mNADH-dependent color production, and (5) digital imaging of colonies to identify mNAD+-active mutants. This method was used to improve the activity of 6-phosphogluconate dehydrogenase on nicotinamide mononucleotide (NMN+). The best mutant obtained after six rounds of directed evolution exhibits a 50-fold enhancement in catalytic efficiency (kcat/KM) and a specific activity of 17.7 U/mg on NMN+, which is comparable to the wild-type enzyme on its natural coenzyme, NADP+. The engineered dehydrogenase was then used to construct an NMNH regeneration system to drive an ene-reductase catalysis. A comparable level of turnover frequency and product yield was observed using the engineered system relative to NADPH regeneration by using the wild-type dehydrogenase. NESPA provides a simple and accurate readout of mNAD+-based activities and the screening at high-throughput levels (approximately tens of thousands per round), thus opening up an avenue for the evolution of dehydrogenases with specific activities on mNAD+s similar to the levels of natural enzyme/coenzyme pairs. Graphical Abstract

SUBMITTER: Huang R

PROVIDER: S-EPMC8580058 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:One of the biochemical abnormalities found in diabetic tissues is a decrease in the cytosolic oxidized to reduced forms of the nicotinamide adenine dinucleotide ratio (NAD+ /NADH also known as pseudohypoxia) caused by oxidation of excessive substrates (glucose through the polyol pathway, free fatty acids and lactate). Subsequently, a decline in NAD+ levels as a result of the activation of poly adenine nucleotide diphosphate-ribose polymerase (mainly in type 1 diabetes) or the inhibition of adenine nucleotide monophosphate-activated protein kinase (in type 2 diabetes). Thus, replenishment of NAD+ levels by nicotinamide-related compounds could be beneficial. However, these compounds also increase nicotinamide catabolites that cause oxidative stress. This is particularly troublesome for patients with diabetes, because they have impaired nicotinamide salvage pathway reactions at the level of nicotinamide phosphoribosyl transferase and phosphoribosyl pyrophosphate, which occurs by the following mechanisms. First, phosphoribosyl pyrophosphate synthesis from pentose phosphate pathway is compromised by a decrease in plasma thiamine and transketolase activity. Second, nicotinamide phosphoribosyl transferase expression is decreased because of reduced adenosine monophosphate-activated protein kinase activity, which occurs in type 2 diabetes. The adenosine monophosphate-activated protein kinase inhibition is caused by an activation of protein kinase C and D1 as a result of enhanced diacylglycerol synthesis caused by pseudohypoxia and increased fatty acids levels. In this regard, nicotinamide-related compounds should be given with caution to treat diabetes. To minimize the risk and maximize the benefit, nicotinamide-related compounds should be taken with insulin sensitizers (for type 2 diabetes), polyphenols, benfotiamine, acetyl-L-carnitine and aldose reductase inhibitors. The efficacy of these regimens can be monitored by measuring serum NAD+ and urinary nicotinamide catabolites.

Project description:BackgroundIt is well known that muscle disuse atrophy is associated with mitochondrial dysfunction, which is implicated in reduced nicotinamide adenine dinucleotide (NAD+ ) levels. Nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in NAD+ biosynthesis, may serve as a novel strategy to treat muscle disuse atrophy by reversing mitochondrial dysfunction.MethodsTo investigate the effects of NAMPT on the prevention of disuse atrophy of skeletal muscles predominantly composed of slow-twitch (type I) or fast-twitch (type II) fibres, rabbit models of rotator cuff tear-induced supraspinatus muscle atrophy and anterior cruciate ligament (ACL) transection-induced extensor digitorum longus (EDL) atrophy were established and then administered NAMPT therapy. Muscle mass, fibre cross-sectional area (CSA), fibre type, fatty infiltration, western blot, and mitochondrial function were assayed to analyse the effects and molecular mechanisms of NAMPT in preventing muscle disuse atrophy.ResultsAcute disuse of the supraspinatus muscle exhibited significant loss of mass (8.86 ± 0.25 to 5.10 ± 0.79 g; P < 0.001) and decreased fibre CSA (3939.6 ± 136.1 to 2773.4 ± 217.6 μm2 , P < 0.001), which were reversed by NAMPT (muscle mass 6.17 ± 0.54 g, P = 0.0033; fibre CSA, 3219.8 ± 289.4 μm2 , P = 0.0018). Disuse-induced impairment of mitochondrial function were significantly improved by NAMPT, including citrate synthase activity (40.8 ± 6.3 to 50.5 ± 5.6 nmol/min/mg, P = 0.0043), and NAD+ biosynthesis (279.9 ± 48.7 to 392.2 ± 43.2 pmol/mg, P = 0.0023). Western blot revealed that NAMPT increases NAD+ levels by activating NAMPT-dependent NAD+ salvage synthesis pathway. In supraspinatus muscle atrophy due to chronic disuse, a combination of NAMPT injection and repair surgery was more effective than repair in reversing muscle atrophy. Although the predominant composition of EDL muscle is fast-twitch (type II) fibre type that differ from supraspinatus muscle, its mitochondrial function and NAD+ levels are also susceptible to disuse. Similar to the supraspinatus muscle, NAMPT-elevated NAD+ biosynthesis was also efficient in preventing EDL disuse atrophy by reversing mitochondrial dysfunction.ConclusionsNAMPT-elevated NAD+ biosynthesis can prevent disuse atrophy of skeletal muscles that predominantly composed with either slow-twitch (type I) or fast-twitch (type II) fibres by reversing mitochondrial dysfunction.

Dataset Information

A High-Throughput Method for Directed Evolution of NAD(P)+-Dependent Dehydrogenases for the Reduction of Biomimetic Nicotinamide Analogues

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets