Expression, biochemical characterization, and mutation of a water forming NADH: FMN oxidoreductase from Lactobacillus rhamnosus

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Highlights

  • A novel water-forming flavin oxidoreductase was first expressed and characterized.

  • A rate-limiting Thr29 was identified and confirmed to be employed to improve its activity.

  • The distance between FMN and NADH was shortened by introducing a small side chain containing residue into position 29.

Abstract

Enzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H2O2 limits its further applications at the industrial scale. Here, novel NADH: FMN oxidoreductase (LrFOR) from Lactobacillus rhamnosus comprised of 1146 bp with a predicted molecular weight of 42 kDa was cloned and overexpressed in Escherichia coli BL21 (DE3). Enzyme assay shows that the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. Biochemical characterizations suggested that LrFOR exhibits the specific activity of 39.8 U·mg−1 with the optimal pH and temperature of 5.6 and 35 °C and produces H2O instead of potentially harmful peroxide. To further study its catalytic function, a critical Thr29 residue and its six mutants were investigated. Mutants T29G, T29A, and T29D show notable enhancement in activities compared with the wild type. Molecular docking of NADH into wild type and its mutants reveal that a small size or electronegative of residue in position29 could shorten the distance of NADH and FMN, promoting the electrons transfer and resulting in the increased activity. This work reveals the pivotal role of position 29 in the catalytic function of LrFOR and provides effective catalysts in NAD+ regeneration.

Introduction

Nicotinamide cofactors (NAD(P)H and NAD(P)+) are important cofactors that act in plenty of redox reactions and regulates the metabolic balance in various genetic processes [[1], [2], [3]]. In industries, they are primary cofactors for many oxidoreductases in the biosynthesis of critical chiral intermediates [[4], [5], [6], [7]]. To prevent the equimolar amount of consumption of these expensive cofactors, it is highly necessary to regenerate them with an efficient and environment-friendly regeneration system [8,9]. Previously, enzymatic, chemical, electrochemical, photochemical, and biological approaches have been proposed for cofactors regeneration [4,5]. The most promising method is the regeneration system of an accessorial redox enzyme with the consumption of coenzyme [[10], [11], [12], [13], [14]].

Thus far, the regeneration of reduced nicotinamide cofactors (NAD(P)H) was well established and these enzymatic methods for NADH regeneration contain the regeneration systems with formate dehydrogenase [15], phosphate dehydrogenase [16], or glucose dehydrogenase [17]. However, the recycling of oxidized counterparts (NAD(P)+) remains a challenging task [1,10]. Different enzymatic methods had been proposed to achieve the regeneration of NAD(P)+. Examples include glutamate dehydrogenase with 2-ketoglutarate [18], alcohol dehydrogenase with acetaldehyde and lactate dehydrogenase with the pyruvate [19]. However, these strategies used for the NAD(P)+ regeneration require the addition of stoichiometric co-substrate, which may complicate the purification process. The procedure for NAD(P)+ regeneration generally considered best is that using NADH: NADH: FMN oxidoreductases with only dioxygen as the ultimate oxidizing agent because of the economic costs of reaction could be reduced obviously.

NADH: FMN oxidoreductases (FOR) are old yellow enzyme members with a (β/α) 8-barrel structure and can catalyze the oxidation of NADH to NAD+ with the flavin mononucleotide (FMN) functions as the prosthetic group [20,21]. In the first step, a semiquinone intermediate (FMNH) was formed by the transfer of the hydride from the nicotinamide group of NADH to the N5 in the isoalloxazine moiety of the oxidized FMN. Then, a proton transfer to the N atom near the ribitol moiety of FMNH which may result in the formation of FMNH2. Eventually, the reduced FMNH2 was oxidized to FMN by O2 molecules (Fig. 1). Several NADH: FMN oxidoreductases had been isolated and identified in various bacteria such as Entamoeba histolytica [22], thermophile Geobacillus stearothermophilus [21], Rhodococcus erythropolis [23] and Methanococcus vannielii [24]. However, all these reported enzymes generate H2O2 as a by-product which even in small amounts can deactivate the enzymes of the cofactor–regeneration reaction. And the addition of catalase as a possible remedy also increases the complexity of the reaction system. Water forming NADH: FMN oxidoreductases attract much attention because the dioxygen was ultimately converted to water rather than harmful hydrogen peroxide. Thus, it is urgent to find a novel H2O-forming FOR to facilitate the regeneration of oxidized counterparts (NAD(P)+) at the industrial scale.

In the present work, a novel H2O-forming NADH: FMN oxidoreductase with dual substrate specificity (NADH and NADPH) from Lactobacillus rhamnosus (LrFOR) was firstly identified and expressed successfully. The characterized LrFOR is stable and active in room temperature and acidulous conditions. By analyzing the sequence and structure information, rational engineering of LrFOR was carried out. Several variants show enhanced activity towards NADH. And the docking of NADH into mutant proteins was used to explore the structural basis behind its improved activity.

Section snippets

Chemicals and bacterial strains

Kanamycin sulfate, isopropyl β-d-thiogalactopyranoside (IPTG), β-nicotinamide adenine dinucleotide reduced dipotassium salt (NADH), 2,2-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid) (ABTS) and horseradish peroxidase (HRP) were purchased from Aladdin (Shanghai, China). Flavin Mononucleotide (FMN) and dithiothreitol (DTT) were purchased from TCI (Tokyo, Japan). Dihydronicotinamide adenine dinucleotide phosphate tetrasodium salt (NADPH) was purchased from Bioduly (Nanjing, China). The

Heterologous expression and purification of LrFOR

A search of the whole genome of L. rhamnosus ATCC 53103 in Genbank database (accession number 6420447) revealed the presence of an NADH: FMN oxidoreductase. The LrFOR gene encodes a polypeptide of 381 amino acids, and it has a calculated molecular mass of 42 kDa. The gene was amplified by PCR using gene-specific primers that amplified a DNA fragment of 1146 nucleotides that represents the full-length NADH: FMN oxidoreductase gene. Then the LrFOR gene was sub-cloned into the expression vector

Discussion

Oxidized nicotinamide cofactors (NAD(P)+) are often required in biocatalytic oxidoreduction, which is significant in enantiopure chemicals and pharmaceutical industries. Enzymatic cofactor regeneration systems are a practical approach to avoid the use of expensive reagents [37]. Compared with most existing NAD(P)+ enzymes which require the addition of cosubstrate, NADH: FMN oxidoreductases only use oxygen as substrate and thus show higher potential for cofactor regeneration.

However, research

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgment

This work was supported by the National Natural Science Foundation of China (No. 21376110).

References (47)

  • J. Pontius et al.

    Deviations from standard atomic volumes as a quality measure for protein crystal structures

    J. Mol. Biol.

    (1996)
  • N. Sreerama et al.

    A self-consistent method for the analysis of protein secondary structure from circular dichroism

    Anal. Biochem.

    (1993)
  • R.A. Sheldon

    9.15 Industrial applications of asymmetric synthesis using cross-linked enzyme aggregates

    Compr. Chirality

    (2012)
  • H.S. Lo et al.

    Purification and properties of NADPH: flavin oxidoreductase from Entamoeba histolytica

    Mol. Biochem. Parasitol.

    (1980)
  • Y. Yu

    Significant improvement of oxidase activity through the genetic incorporation of a redox-active unnatural amino acid

    Chem. Sci.

    (2015)
  • P.B. Brondani et al.

    Finding the switch: turning a baeyer-villiger monooxygenase into a NADPH oxidase

    J. Am. Chem. Soc.

    (2014)
  • K.W. Ryu et al.

    Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis

    Science

    (2018)
  • Z. Xiao et al.

    A novel whole-cell biocatalyst with NAD+ regeneration for production of chiral chemicals

    PLoS One

    (2010)
  • M.Y. Zhuang et al.

    Immobilization of glycerol dehydrogenase and NADH oxidase for enzymatic synthesis of 1,3-dihydroxyacetone with in situcofactor regeneration: enzymatic production of 1, 3-dihydroxyacetone via immobilized enzymes

    J. Chem. Technol. Biotechnol.

    (2018)
  • M. Leonida

    Redox enzymes used in chiral syntheses coupled to coenzyme regeneration

    Curr. Med. Chem.

    (2001)
  • C.H. Liu et al.

    Preparation of functionalized graphene oxide nanocomposites for covalent immobilization of NADH oxidase

    Nanosci. Nanotechnol. Lett.

    (2016)
  • Y. Wen et al.

    Discovery of a novel inhibitor of NAD(P)+-dependent malic enzyme (ME2) by high-throughput screening

    Acta Pharmacol. Sin.

    (2016)
  • H. Gao et al.

    Role of surface residue 184 in the catalytic activity of NADH oxidase from Streptococcus pyogenes

    Appl. Microbiol. Biotechnol.

    (2014)
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