Change of Nucleotide Specificity and Enhancement of Catalytic Efficiency in Single Point Mutants ofVibrio harveyiAldehyde Dehydrogenase†

Ala-114, together with Asp-113, Tyr-115 and Gln-151, form the pocket that accommodates the 3′-OH of the incoming dNTP in the HIV-1 RT (reverse transcriptase). Four mutant RTs having serine, glycine, threonine or valine instead of Ala-114 were obtained by site-directed mutagenesis. While mutants A114S and A114G retained significant DNA polymerase activity, A114T and A114V showed very low catalytic efficiency in nucleotide incorporation assays, due to their high apparent Km values for dNTP. Discrimination between AZTTP (3′-azido-3′-deoxythymidine triphosphate) and dTTP was not significantly affected by mutations A114S and A114G in assays carried out with heteropolymeric template/primers. However, both mutants showed decreased susceptibility to AZTTP when poly(rA)/(dT)16 was used as substrate. Steady-state kinetic analysis of the incorporation of ddNTPs compared with dNTPs showed that substituting glycine for Ala-114 produced a 5–6-fold increase in the RT's ability to discriminate against ddNTPs (including the physiologically relevant metabolites of zalcitabine and didanosine), a result that was confirmed in primer-extension assays. In contrast, A114S and A114V showed wild-type ddNTP/dNTP discrimination efficiencies. Discrimination against ribonucleotides was not affected by mutations at position 114. Misinsertion and mispair extension fidelity assays as well as determinations of G→A mutation frequencies using a lacZ complementation assay showed that, unlike Tyr-115 or Gln-151 mutants, the fidelity of HIV-1 RT was not largely affected by substitutions of Ala-114. The role of the side-chain of Ala-114 in ddNTP/dNTP discrimination appears to be determined by its participation in van der Waals interactions with the ribose moiety of the incoming nucleotide.

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Reduction of The Pyruvate Decarboxylase Activity Improves Isobutanol Production By Klebsiella Pneumoniae

10.21203/rs.3.rs-1155955/v1 ◽

2021 ◽

Author(s):

Lin Shu ◽

Jinjie Gu ◽

Qinghui Wang ◽

Shaoqi Sun ◽

Youtian Cui ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Single Point ◽

Pyruvate Decarboxylase ◽

Catalytic Efficiency ◽

Decarboxylase Activity ◽

Enzymatic Reactions ◽

Substrate Conversion ◽

High Level

Abstract Background Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. ipdC, annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate. Results Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis, Kp-IpdC has an 2.8-fold lower Km for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, high level expression of ipdC by induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling we identified 10 residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-mutants were selected for exploration. Mutants L546W and T290L showed 5.1% and 22.1% of catalytic efficiency on pyruvate, which were then expressed in K. pneumoniae for in vivo test. Isobutanol production by K. pneumoniae T290L was 25% higher than the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose. Conclusions This research provides a new way to improve the efficiency of the biological route of isobutanol production.

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Enzymatic Dynamic Reductive Kinetic Resolution Towards 115 g/L (S)-2-Phenylpropanol

10.21203/rs.3.rs-183141/v1 ◽

2021 ◽

Author(s):

Christian Rapp ◽

Simone Pival-Marko ◽

Erika Tassano ◽

Bernd Nidetzky ◽

Regina Kratzer

Keyword(s):

Kinetic Resolution ◽

Single Point ◽

Xylose Reductase ◽

Catalytic Efficiency ◽

Dry Weight ◽

Catalyst Loading ◽

A Value ◽

Whole Cell Biocatalyst ◽

Very High

Abstract BackgroundPublished biocatalytic routes towards chiral 2-phenylpropanol by oxidoreductases showed product concentrations of maximally 80 mM. Enzyme deactivation turned out as one major limitation and was attributed to adduct formation of the aldehyde substrate with the catalytic reductase.ResultsA Candida tenuis xylose reductase single-point mutant (CtXR D51A) with very high catalytic efficiency (43·103 s-1M-1) for (S)-2-phenylpropanal was identified. The enzyme showed high enantioselectivity for the (S)-enantiomer but was deactivated by 0.5 mM substrate within 2 h. A whole-cell biocatalyst based on the engineered reductase and a yeast formate dehydrogenase for NADH-recycling provided substantial stabilization of the reductase. The relatively slow in situ racemization of 2-phenylpropanal and the still limited biocatalyst stability required a subtle adjustment of the substrate-to-catalyst ratio. A value of 3.4 gsubstrate/gcell-dry-weight turned out as compromise between product enantiopurity and conversion. A catalyst loading of 40 gcell-dry-weight was used to convert 1 M racemic 2-phenylpropanal to (S)-phenylpropanol in 93.1 % e.e. ConclusionMainly hydrolases have been exploited for the production of profenols at industrial scale so far. The herein established bioreduction presents an alternative route towards profenols that is competitive to hydrolase-catalyzed kinetic resolutions.

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Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

10.26434/chemrxiv.13311614 ◽

2020 ◽

Author(s):

Piia Kokkonen ◽

Andy Beier ◽

Stanislav Mazurenko ◽

Jiri Damborsky ◽

David Bednar ◽

...

Keyword(s):

Single Point ◽

Substrate Inhibition ◽

Catalytic Efficiency ◽

Single Point Mutation ◽

Substrate Complex ◽

Enzyme Product ◽

Enzyme Substrate ◽

Markov State Models ◽

State Models ◽

Dynamics Simulations

<div> <p>Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme-product complex. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. These results show that substrate inhibition can be caused by substrate binding to the enzyme-product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels. </p> </div> <br>

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TRPM7 Channel Is Regulated by Magnesium Nucleotides via its Kinase Domain

The Journal of General Physiology ◽

10.1085/jgp.200509410 ◽

2006 ◽

Vol 127 (4) ◽

pp. 421-434 ◽

Cited By ~ 128

Author(s):

Philippe Demeuse ◽

Reinhold Penner ◽

Andrea Fleig

Keyword(s):

Binding Site ◽

Single Point ◽

Energy Levels ◽

Nucleotide Binding ◽

Kinase Domain ◽

Channel Activity ◽

Protein Kinase Domain ◽

Functional Roles ◽

Cell Energy ◽

Nucleotide Specificity

TRPM7 is a Ca2+- and Mg2+-permeable cation channel that also contains a protein kinase domain. While there is general consensus that the channel is inhibited by free intracellular Mg2+, the functional roles of intracellular levels of Mg·ATP and the kinase domain in regulating TRPM7 channel activity have been discussed controversially. To obtain insight into these issues, we have determined the effect of purine and pyrimidine magnesium nucleotides on TRPM7 currents and investigated the possible involvement of the channel's kinase domain in mediating them. We report here that physiological Mg·ATP concentrations can inhibit TRPM7 channels and strongly enhance the channel blocking efficacy of free Mg2+. Mg·ADP, but not AMP, had similar, albeit smaller effects, indicating a double protection against possible Mg2+ and Ca2+ overflow during variations of cell energy levels. Furthermore, nearly all Mg-nucleotides were able to inhibit TRPM7 activity to varying degrees with the following rank in potency: ATP > TTP > CTP ≥ GTP ≥ UTP > ITP ≈ free Mg2+ alone. These nucleotides also enhanced TRPM7 inhibition by free Mg2+, suggesting the presence of two interacting binding sites that jointly regulate TRPM7 channel activity. Finally, the nucleotide-mediated inhibition was lost in phosphotransferase-deficient single-point mutants of TRPM7, while the Mg2+-dependent regulation was retained with reduced efficacy. Interestingly, truncated mutant channels with a complete deletion of the kinase domain regained Mg·NTP sensitivity; however, this inhibition did not discriminate between nucleotide species, suggesting that the COOH-terminal truncation exposes the previously inaccessible Mg2+ binding site to Mg-nucleotide binding without imparting nucleotide specificity. We conclude that the nucleotide-dependent regulation of TRPM7 is mediated by the nucleotide binding site on the channel's endogenous kinase domain and interacts synergistically with a Mg2+ binding site extrinsic to that domain.

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An approach to optimizing the active site in a glutathione transferase by evolution in vitro

Biochemical Journal ◽

10.1042/bj3440093 ◽

1999 ◽

Vol 344 (1) ◽

pp. 93-100 ◽

Cited By ~ 5

Author(s):

Lars O. HANSSON ◽

Mikael WIDERSTEN ◽

Bengt MANNERVIK

Keyword(s):

Active Site ◽

Glutathione Transferase ◽

Single Point ◽

Active Form ◽

Catalytic Efficiency ◽

Order Rate Constant ◽

Single Point Mutation ◽

Transition State Analogue ◽

Active Site Mutants

A glutathione transferase (GST) mutant with four active-site substitutions (Phe10 → Pro/Ala12 → Trp/Leu107 → Phe/Leu108 → Arg) (C36) was isolated from a library of active-site mutants of human GST A1-1 by the combination of phage display and mechanism-based affinity adsorption [Hansson, Widersten and Mannervik (1997) Biochemistry 36, 11252-11260]. C36 was selected on the basis of its affinity for the transition-state analogue 1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate. C36 affords a 105-fold rate enhancement over the uncatalysed reaction between reduced glutathione and 1-chloro-2,4-dinitrobenzene (CDNB), as evidenced by the ratio between kcat/Km and the second-order rate constant k2. The present study shows that C36 can evolve to an even higher catalytic efficiency by an additional site-specific mutation. Random mutations of the fifth active-site residue 208 allowed the identification of 18 variants, of which the mutant C36 Met208 → Cys proved to be the most active form. The altered activity was substrate selective such that the catalytic efficiency with CDNB and with 1-chloro-6-trifluoromethyl-2,4-dinitrobenzene were increased 2-3-fold, whereas the activity with ethacrynic acid was decreased by a factor of 8. The results show that a single-point mutation in the active site of an enzyme may modulate the catalytic activity without being directly involved as a functional group in the enzymic mechanism. Such limited modifications are relevant both to the natural evolution and the in vitro redesign of proteins for novel functions.

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Alcohol discrimination and preferences in two species of nectar-feeding primate

Royal Society Open Science ◽

10.1098/rsos.160217 ◽

2016 ◽

Vol 3 (7) ◽

pp. 160217 ◽

Cited By ~ 7

Author(s):

Samuel R. Gochman ◽

Michael B. Brown ◽

Nathaniel J. Dominy

Keyword(s):

Single Point ◽

Catalytic Efficiency ◽

Selective Advantage ◽

Alcohol Concentration ◽

Single Point Mutation ◽

Fermented Foods ◽

Last Common Ancestor ◽

Slow Loris ◽

Nectar Feeding ◽

Nycticebus Coucang

Recent reports suggest that dietary ethanol, or alcohol, is a supplemental source of calories for some primates. For example, slow lorises ( Nycticebus coucang ) consume fermented nectars with a mean alcohol concentration of 0.6% (range: 0.0–3.8%). A similar behaviour is hypothesized for aye-ayes ( Daubentonia madagascariensis ) based on a single point mutation (A294V) in the gene that encodes alcohol dehydrogenase class IV (ADH4), the first enzyme to catabolize alcohol during digestion. The mutation increases catalytic efficiency 40-fold and may confer a selective advantage to aye-ayes that consume the nectar of Ravenala madagascariensis . It is uncertain, however, whether alcohol exists in this nectar or whether alcohol is preferred or merely tolerated by nectarivorous primates. Here, we report the results of a multiple-choice food preference experiment with two aye-ayes and a slow loris. We conducted observer-blind trials with randomized, serial dilutions of ethanol (0–5%) in a standard array of nectar-simulating sucrose solutions. We found that both species can discriminate varying concentrations of alcohol; and further, that both species prefer the highest available concentrations. These results bolster the hypothesized adaptive function of the A294V mutation in ADH4, and a connection with fermented foods, both in aye-ayes and the last common ancestor of African apes and humans.

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Substrate Inhibition by the Blockage of Product Release and Its Control by Tunnel Engineering

10.26434/chemrxiv.13311614.v1 ◽

2020 ◽

Author(s):

Piia Kokkonen ◽

Andy Beier ◽

Stanislav Mazurenko ◽

Jiri Damborsky ◽

David Bednar ◽

...

Keyword(s):

Single Point ◽

Substrate Inhibition ◽

Catalytic Efficiency ◽

Single Point Mutation ◽

Substrate Complex ◽

Enzyme Product ◽

Enzyme Substrate ◽

Markov State Models ◽

State Models ◽

Dynamics Simulations

<div> <p>Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition. Surprisingly, a global kinetic analysis suggested that this inhibition is caused by binding of the substrate to the enzyme-product complex. Molecular dynamics simulations clarified the details of this unusual mechanism of substrate inhibition: Markov state models indicated that the substrate prevents the exit of the halide product by direct blockage and/or restricting conformational flexibility. The contributions of three residues forming the possible substrate inhibition site (W140A, F143L and I211L) to the observed inhibition were studied by mutagenesis. An unusual synergy giving rise to high catalytic efficiency and reduced substrate inhibition was observed between residues L177W and I211L, which are located in different access tunnels of the protein. These results show that substrate inhibition can be caused by substrate binding to the enzyme-product complex and can be controlled rationally by targeted amino acid substitutions in enzyme access tunnels. </p> </div> <br>

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Mutagenesis of Glycine 179 Modulates Both Catalytic Efficiency and Reduced Pyridine Nucleotide Specificity in Cytochromeb5Reductase†

Biochemistry ◽

10.1021/bi051165t ◽

2005 ◽

Vol 44 (41) ◽

pp. 13467-13476 ◽

Cited By ~ 11

Author(s):

Glenn W. Roma ◽

Louis J. Crowley ◽

C. Ainsley Davis ◽

Michael J. Barber

Keyword(s):

Catalytic Efficiency ◽

Pyridine Nucleotide ◽

Nucleotide Specificity

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Reductive enzymatic dynamic kinetic resolution affording 115 g/L (S)-2-phenylpropanol

BMC Biotechnology ◽

10.1186/s12896-021-00715-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Christian Rapp ◽

Simone Pival-Marko ◽

Erika Tassano ◽

Bernd Nidetzky ◽

Regina Kratzer

Keyword(s):

Kinetic Resolution ◽

Single Point ◽

Xylose Reductase ◽

Catalytic Efficiency ◽

Dry Weight ◽

Catalyst Loading ◽

Amino Acid Residues ◽

A Value ◽

Very High

Abstract Background Published biocatalytic routes for accessing enantiopure 2-phenylpropanol using oxidoreductases afforded maximal product titers of only 80 mM. Enzyme deactivation was identified as the major limitation and was attributed to adduct formation of the aldehyde substrate with amino acid residues of the reductase. Results A single point mutant of Candida tenuis xylose reductase (CtXR D51A) with very high catalytic efficiency (43·103 s−1 M−1) for (S)-2-phenylpropanal was found. The enzyme showed high enantioselectivity for the (S)-enantiomer but was deactivated by 0.5 mM substrate within 2 h. A whole-cell biocatalyst expressing the engineered reductase and a yeast formate dehydrogenase for NADH-recycling provided substantial stabilization of the reductase. The relatively slow in situ racemization of 2-phenylpropanal and the still limited biocatalyst stability required a subtle adjustment of the substrate-to-catalyst ratio. A value of 3.4 gsubstrate/gcell-dry-weight was selected as a suitable compromise between product ee and the conversion ratio. A catalyst loading of 40 gcell-dry-weight was used to convert 1 M racemic 2-phenylpropanal into 843 mM (115 g/L) (S)-phenylpropanol with 93.1% ee. Conclusion The current industrial production of profenols mainly relies on hydrolases. The bioreduction route established here represents an alternative method for the production of profenols that is competitive with hydrolase-catalyzed kinetic resolutions.

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