TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH

Nisar, Muhammad Atif; Rashid, Naeem; Bashir, Qamar; Gardner, Qurratulann Afza; Shafiq, Muhammad; Akhtar, Muhammad

doi:10.1016/j.jbiosc.2013.01.020

Cited by 13 publications

(15 citation statements)

References 14 publications

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“…Sequence comparison of H 2 O 2 and H 2 O forming NAD(P)H oxidase from T. kodakarensis ( Tk NOX) and Sm NOX showed that Tk NOX features an arginine at positions equivalent to Sm NOX positions 194 and 199 and a lysine at the position equivalent to Sm NOX position 200 [45] (Figure 1). The results from the Sm NOX mutation study indicate that these positively charged residues might be responsible for making Tk NOX the only known bacterial wild type NOX showing higher activity with NADPH than with NADH.…”

Section: Resultsmentioning

confidence: 99%

“…Water-forming NADH oxidases have been studied from several bacteria as Streptococcus [32–35], Lactobacillus [36–38], Lactococcus [39], Clostridium [40], Serpulina [41], Leuconostoc [42] and Bacillus [43]. NOX from P. furiosus [44] and T. kodakarensis [45] produce H 2 O and a significant level of H 2 O 2 (72% for Pf NOX, 25% for Tk NOX). NAD(P)H oxidases belong to the pyridine nucleotide disulfide oxidoreductases (PNDOR) together with, among others, glutathione reductase and CoA-disulfide reductase [46].…”

Section: Introductionmentioning

confidence: 99%

“…Overoxidation of the cysteine leads to enzyme deactivation. Several NADH oxidases need FADH or DTT addition for optimal performance [36, 45, 47]. Enzymes with high specific activities (> 150 U/mg) were recently reported from L. sanfranciscensis, L. plantarum , L. rhamnosus and S. pyogenes [34, 37, 38, 48].…”

Section: Introductionmentioning

confidence: 99%

“…In wild type form only two water forming NOXs and one hydrogen peroxide forming NOX show activity with NADPH (around half / one third of activity with NADH depending on the NOX [38, 49, 50]. NOX 1299 from T. kodakarensis is the only NOX showing higher wild type activity with NADPH than with NADH but disadvantageously it produces high amounts of H 2 O 2 [45]. NOX of L. plantarum was recently successfully mutated to accept NADPH but resulting in a simultaneous decrease in NADH activity [37].…”

Section: Introductionmentioning

confidence: 99%

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Cofactor Specificity Engineering of Streptococcus Mutans Nadh Oxidase 2 for Nad(p) + Regeneration in Biocatalytic Oxidations

Petschacher

Staunig

Müller

et al. 2014

Computational and Structural Biotechnology Journal

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Soluble water-forming NAD(P)H oxidases constitute a promising NAD(P)+ regeneration method as they only need oxygen as cosubstrate and produce water as sole byproduct. Moreover, the thermodynamic equilibrium of O2 reduction is a valuable driving force for mostly energetically unfavorable biocatalytic oxidations. Here, we present the generation of an NAD(P)H oxidase with high activity for both cofactors, NADH and NADPH. Starting from the strictly NADH specific water-forming Streptococcus mutans NADH oxidase 2 several rationally designed cofactor binding site mutants were created and kinetic values for NADH and NADPH conversion were determined. Double mutant 193R194H showed comparable high rates and low K m values for NADPH (k cat 20 s-1, K m 6 µM) and NADH (k cat 25 s-1, K m 9 µM) with retention of 70% of wild type activity towards NADH. Moreover, by screening of a SeSaM library S. mutans NADH oxidase 2 variants showing predominantly NADPH activity were found, giving further insight into cofactor binding site architecture. Applicability for cofactor regeneration is shown for coupling with alcohol dehydrogenase from Sphyngobium yanoikuyae for 2-heptanone production.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cofactor Specificity Engineering of Streptococcus Mutans Nadh Oxidase 2 for Nad(p) + Regeneration in Biocatalytic Oxidations

Petschacher

Staunig

Müller

et al. 2014

Computational and Structural Biotechnology Journal

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“…The activities of G6PDH and 6PGDH are traditionally monitored by following NADPH production spectrophotometrically, but our assays employing total cell extracts were then subject to the competing activities of the strong NADPH-oxidase activity observed in the controls [494]. In an attempt to circumvent these competing activities, the G6PDH and 6PGDH activity assays were repeated anaerobically such that NADPH-oxidase activity was now severely hampered by the lack of oxygen.…”

Section: Confirmation Of Enzyme Activitymentioning

confidence: 99%

Thermococcus kodakarensis : the key to affordable biohydrogen production

Spaans¹

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Characterization and gene deletion analysis of four homologues of group 3 pyridine nucleotide disulfide oxidoreductases from Thermococcus kodakarensis

et al. 2014

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elemental sulfur (S(0)) oxidoreductase (NSR) and NAD(P)H oxidase (NOX) activities. TK1299 exhibited NSR activity with a preference for NADPH and showed strict CoA-dependency similar to that of the Pyrococcus furiosus homologue PF1186. During the assays, the non-enzymatic formation of H2S from S(0) and free CoA-SH was observed, and the addition of enzyme and NADPH enhanced H2S evolution. A catalytic cycle of TK1299 was proposed suggesting that CoA-SH acted to solubilize S(0) by forming CoA persulfides, followed by reduction of an enzyme-S-S-CoA intermediate produced after both enzymatic and non-enzymatic evolution of H2S from the CoA persulfide, with NADPH as an electron donor. TK1481 showed NSR activity independently of CoA-SH, implying a direct reaction with S(0). TK1299, TK1481, and TK0304 exhibited high NOX activity, and the NADH-dependent activities were inhibited by the addition of free CoA-SH. Multiple disruptions of the four group 3 PNDOR homologues in T. kodakarensis demonstrated that none of these homologues were essential for S(0)-dependent growth. Many disruptants grew better than the parent strain, but a few multiple disruptants showed decreased growth properties after aerobic inoculation into a pyruvate-containing medium without S(0), suggesting the complicated participation of these group 3 PNDORs in sensitivity/resistance to dissolved oxygen when S(0) was absent.

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TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH

Cited by 13 publications

References 14 publications

Cofactor Specificity Engineering of Streptococcus Mutans Nadh Oxidase 2 for Nad(p) + Regeneration in Biocatalytic Oxidations

Cofactor Specificity Engineering of Streptococcus Mutans Nadh Oxidase 2 for Nad(p) + Regeneration in Biocatalytic Oxidations

Thermococcus kodakarensis : the key to affordable biohydrogen production

Characterization and gene deletion analysis of four homologues of group 3 pyridine nucleotide disulfide oxidoreductases from Thermococcus kodakarensis

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