Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993
and Svetlana Tishchenko 1Round 1
Reviewer 1 Report
The presented manuscript describes a set of mutant forms of 2D laccase from Streptomyces griseoflavus. The strong point of the work is the presence of structural data as well as reported biochemical properties for these mutant forms. The presented data are novel and significance of content is high. However, the structure-function analysis performed has several flaws. My main concern is about the reported Cu1 concentration measurements using UV-Vis spectra and the further conclusions regarding laccase thermo inactivation. See comment for L201-207. Discussion section is very long and contains a lot of speculative parts. I recommend carefully revise and shorten this section.
I also ask to provide the access to structural data.
L10: “Laccases catalyze the oxidation of substrates…” – please, add the exact types of substrates.
L32: Please, replace Ref.1 to something more appropriate; please, rephrase the part “Due to low substrate specificity…”. It sounds a little bit confusing.
L66: In p-toluate there are no atoms which can de oxidized by laccase. Please, correct this part.
L84-85: The statement is not confirmed by the literature cited above. Please, add more relevant references.
L97-98: What do you mean by “activity rate”?
L116 (Figure 1): The position of the discussed aa residues is not clear from the figure. I recommend showing the protein backbone and maybe the neighboring chain. Please, add the PDB ID of the structure used to the figure caption.
L148-149: “Surprisingly, the pH optimum of 148 His165Ala/Met199Gly variant is pH 6.5…” – It is not obvious from the figure. Please, start the axis from pH-values below 6.5.
L155 (Table 2): The scale does not match the color of the table cells.
L183 (Table 3): How do you explain the increase in activity of the His165Ala/Met199Gly mutant after adding of the NaN3?
L188 (Figure 3): Please, correct the axis labels.
L201-207: The performed experiment cannot undoubtedly prove the loss of the copper ions during incubation. The loss of the color could take place because of Cu1 reduction at low concentrations of O2. For investigation of thermal destruction of laccase more advanced techniques should be used like CD-spectrometry, DSC and EPR.
How the protein aggregation was estimated?
L212: Please, indicate the pH of buffer for these experiments. What was the rationale for choosing it?
L232 (Figure6): The panel (a) and panel (b) are shown from different angles, which makes it impossible to compare them. Please, improve the figure.
L282-283: Speculative statement. Are there any other evidences of the increased mobility of the loops other than the second conformations of the side chains of the two aa residues?
L289: The reference to Figure 2 is incorrect.
L291-292: Another questionable statement. Thermal stability is not the indicator of the mobility of the loops.
L295 (Figure 7): The panels (a) and (b) are not cited anywhere in the manuscript. There is also a mistake in the caption – it is a repetition of the caption to figure 6. Also Cu1 coordination bonds are missed at panel (a).
L331-340: There is a long discussion concerning the role His 165 proposed by the authors in the previous publication. However, it seems like mutations of Met199 affects the pH-dependence (especially the residual activity at 6.5) much more than other single mutations. Taking this to account, the assignment the role of pH-optimum affecter to His165 and electron flux enhancer – to Met199 is not as clear as it is presented. This part should be carefully revised.
L342-L343: The reference is missed.
L475: How long the enzyme samples were incubated with NaN3?
Author Response
Answers to referee
The presented manuscript describes a set of mutant forms of 2D laccase from Streptomyces griseoflavus. The strong point of the work is the presence of structural data as well as reported biochemical properties for these mutant forms. The presented data are novel and significance of content is high. However, the structure-function analysis performed has several flaws. My main concern is about the reported Cu1 concentration measurements using UV-Vis spectra and the further conclusions regarding laccase thermo inactivation. See comment for L201-207. Discussion section is very long and contains a lot of speculative parts. I recommend carefully revise and shorten this section.
I also ask to provide the access to structural data.
Dear reviewer, we send the structural data to the editorial office.
L10: “Laccases catalyze the oxidation of substrates…” – please, add the exact types of substrates.
The abstract is limited by 200 words. The exact types of substrates you can find in the first sentence of the Introduction (L29).
“Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyze the oxidation of various organic and inorganic molecules like mono- and diphenols, polyphenols, diamines, aminophenols, aromatic or aliphatic amines with the four-electron reduction of molecular oxygen to water [1,2]”
L32: Please, replace Ref.1 to something more appropriate; please, rephrase the part “Due to low substrate specificity…”. It sounds a little bit confusing.
The Ref. 1 was replaced to “Giardina, P.; Faraco, V.; Pezzella, C.; Piscitelli, A.; Vanhulle, S.; Sannia, G. Laccases: A never-ending story”
We overwrote “Due to low substrate specificity…”
L66: In p-toluate there are no atoms which can de oxidized by laccase. Please, correct this part.
We have removed this sentence.
L84-85: The statement is not confirmed by the literature cited above. Please, add more relevant references.
The additional information and references were added
L97-98: What do you mean by “activity rate”?
The “activity rate” is turnover number (Kcat). It was corrected in the text.
L116 (Figure 1): The position of the discussed aa residues is not clear from the figure. I recommend showing the protein backbone and maybe the neighboring chain. Please, add the PDB ID of the structure used to the figure caption.
Figure 1represent the overall position of Met199, Tyr230, His165, and Arg240 (mutation point) relatively to active center copper ions and tunnels. Protein backbone and the neighboring chain make figure more complicated. We would prefer to leave the Figure 1 as it was. The PDB ID added to the figure caption.
L148-149: “Surprisingly, the pH optimum of His165Ala/Met199Gly variant is pH 6.5…” – It is not obvious from the figure. Please, start the axis from pH-values below 6.5.
Figure 2 was changed appropriately with your recommendation
L155 (Table 2): The scale does not match the color of the table cells.
The colors of the scale-bar of table 2 were corrected.
L183 (Table 3): How do you explain the increase in activity of the His165Ala/Met199Gly mutant after adding of the NaN3?
In our previous article (ref.18) we showed this effect for His165Ala. In this work, for double mutant form His165Ala/Met199Gly, a similar effect after the adding of NaN3 was observed. We have noticed it again, and a detailed study of this effect will be given in the following works. To date, we don't know the exact mechanism of this effect and just wish to emphasize the reproducibility of the result. The crystallographic study of NaN3 binding sites at laccase using crystals of His165Ala and His165Ala/Met199Gly is in progress in our group. This is the topic of our future article
L188 (Figure 3): Please, correct the axis labels.
The axis labels of Figure 3 were corrected.
L201-207: The performed experiment cannot undoubtedly prove the loss of the copper ions during incubation. The loss of the color could take place because of Cu1 reduction at low concentrations of O2. For investigation of thermal destruction of laccase more advanced techniques should be used like CD-spectrometry, DSC and EPR.
The EPR and crystallographic studies of laccases showed that the Cu1 ion reduction occurs only in the case of interaction with substrates, some inhibitors, or electron irradiation [Malmstrom, Mosbach and Vänngård, 1959; Nakamura, 1958; Marta Ferraroni, Nina Myasoedova, Vadim Schmatchenko et al 2007].
The investigation of loss of the copper ions during high-temperature incubation was carried out for both wild type and M199G mutant. The concentration and volume of the samples was similar. The samples were heated up at the same temperature and time range, which means that the decrease in the concentration/amount of oxygen was the same. It is difficult to imagine how the same decrease in oxygen concentration can affect the loss of Cu1 ion in Met199Gly and SgfSL by a different way. In our opinion, the decrease in the intensity of the Сu1 peak upon heating the samples does not depend on the amount of oxygen as the O2 cannot interact with Cu1 ion directly. Besides, we carried out the experiment without substrate as donor electrons. Thereby, no other reason for changing the absorption peak at 600 nm during heating than the loss of a Cu1 ion.
Thermal stability of two-domain laccases unlike 3D laccases is difficult to assess by CD spectroscopy, since the CD spectrum is very weak. Tryptophan fluorescence also does not give convincing results, since the relative absorption curve (315/350nm) does not reach a plateau and it is very difficult to estimate the half-transition temperature (Figure below). We give a graph of the dependence of tryptophan fluorescence on temperature. We used samples of two 2D laccases (SgfSL and SvSL).
Figure . Tryptophan fluorescence curves of 2D laccases Streptomyces griseoflavus (SgfSL, black) and Streptomyces viridochromogenes (SvSL, red).
Unfortunately, we do not have the opportunity to use EPR. However, EPR spectroscopy would allow estimating the amount of copper ion and suggesting its coordination, but the overall stability of the protein is difficult to investigate by these methods. We have used DLS to investigate the protein aggregation after heating. DLS curves showed that laccase samples after the heating did not change (supplementary, Figure. S1).
The goal of this article was to investigate the catalytic activity of SgfSL and mutants after the heating, but not their overall structural ‘‘thermal stability”. In the text we replaced “thermal stability” to “activity of SgfSL after high-temperature incubation”
How the protein aggregation was estimated?
Firstly, the absorption of the samples at 280 nm did not changed during the experiment and precipitation was not observed in SgfSL and Met199Gly samples. Secondly, the absence of protein aggregation confirmed by DLS experiments (supplementary, Figure S1). Light scattering peaks of SgfSL and Met199Gly after heating did not differ (the diameter of the particle close to 10 nm - 70-100kDa).
L212: Please, indicate the pH of buffer for these experiments. What was the rationale for choosing it?
At first, we determined the pH-optimum of dye decoloration by SgfSL and mutant forms with the addition of ABTS as a mediator. The optimum of dyes decolorization (best rate) by Met199Ala and SgfSL was pH=5 and by mutant Met199Gly and Tyr230Ala – pH=6.5. This information was added in the “Materials and Methods”.
L232 (Figure6): The panel (a) and panel (b) are shown from different angles, which makes it impossible to compare them. Please, improve the figure.
The main ideas of the panels are different. Panel (a) represent SPB in detail, whereas panel (b) represents that substrates more massive than 2.6-DMP cannot oxidize simultaneously in the active center of the trimer. A graphical representation of the structures in the same angles, unfortunately, will not be clear due to the residue's side-chain overlapping.
L282-283: Speculative statement. Are there any other evidences of the increased mobility of the loops other than the second conformations of the side chains of the two aa residues?
The certain position of His294 (first coordination sphere of Cu1) is fixed by Met199 and Val291 (Figure 6a). Moreover, the side group of Met199 is additionally fixed with Tyr230. We suggest this system of contacts is responsible for the high stability of the substrate-binding region. The absence of at least one stabilizing element (f.e. the substitution Met199Gly) leads to not only a greater mobility of the other side groups, but also, to a greater mobility of the secondary structural element (long loop).[Imai K, Mitaku S. Mechanisms of secondary structure breakers in soluble proteins].
L289: The reference to Figure 2 is incorrect.
Thank you, the reference was corrected and transformed to Figure 3
L291-292: Another questionable statement. Thermal stability is not the indicator of the mobility of the loops.
Indeed, thermal stability, which denotes the overall structural stability of the protein, is not the indicator of the mobility of the loops.
We showed that the residual activity of SgfSL mutants with replacements close to the loop located in SBP decreased after incubation at a high temperature. No proteins aggregation was observed during the experiment, thereby, we speculate that a possible reason for the decrease in activity may be the loss of Cu1 as a result of the mobility of this loop.
Analysis of the obtained crystal structures showed that the occupancy of Cu1 ions in structures of mutant forms is the same as the wild type laccase. Crystals were growing at room temperature. Spectrophotometric method applied to laccase showed that after heating the Cu1 ion content in mutant forms decrease. We conclude that activity of the enzyme at high temperatures might be associated with the proposed mobility of the loop.
L295 (Figure 7): The panels (a) and (b) are not cited anywhere in the manuscript. There is also a mistake in the caption – it is a repetition of the caption to figure 6. Also Cu1 coordination bonds are missed at panel (a).
Thank you for your attention and comments. We have corrected this mistake and added the citation of missed panels.
L331-340: There is a long discussion concerning the role His 165 proposed by the authors in the previous publication. However, it seems like mutations of Met199 affects the pH-dependence (especially the residual activity at 6.5) much more than other single mutations. Taking this to account, the assignment the role of pH-optimum affecter to His165 and electron flux enhancer – to Met199 is not as clear as it is presented. This part should be carefully revised.
Indeed, strong affects the pH-dependence is manifested on double mutant His165/Met199 but no single mutants. At present time, we investigate this phenomenon using site-direction mutagenesis.
This work in progress and will be the topic of the next article. We would prefer to leave this part of the manuscript as it is.
L342-L343: The reference is missed.
The reference was added.
L475: How long the enzyme samples were incubated with NaN3?
The samples were incubated with sodium azide for 30 seconds before substrate was added. The experiments were also repeated with pre-incubation of the enzymes with sodium azide for 1 minute, and there are no differences were found in comparison with 30 seconds
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The present manuscript dealt with “engineering the catalytic properties of 2D bacterial laccase”. In this study authors constructed, produced/purified, crystallized and investigated the different variants of the 2D laccase from Streptomyces griseoflavus Ac-993. By engineering the SBP of laccase, authors achieved five time increase in the catalytical activity. This is very significant. Authors presented all the data neatly and in standard format. The manuscript is scientifically sound and fits in the scope of the journal. I recommend the minor revisions.
- In introduction section, please emphasize more on broad importance of the laccase SBP domain engineering.
- The mutants showed enhanced pH stabilities than wild type, what is possible explanation for this.
- Does author studied decolorization of dyes without redox mediator ABTS for all mutants and wild type, if mutant exhibit decolorization without redox mediator, it will significant. Also, does any difference in requirement of the redox mediator concentrations in mutant laccases than wild type?
Author Response
The present manuscript dealt with “engineering the catalytic properties of 2D bacterial laccase”. In this study authors constructed, produced/purified, crystallized and investigated the different variants of the 2D laccase from Streptomyces griseoflavus Ac-993. By engineering the SBP of laccase, authors achieved five time increase in the catalytical activity. This is very significant. Authors presented all the data neatly and in standard format. The manuscript is scientifically sound and fits in the scope of the journal. I recommend the minor revisions.
Thank you for your comments.
- In introduction section, please emphasize more on broad importance of the laccase SBP domain engineering.
We have added additional information to the introduction section (lines 79-84, 91-93).
- The mutants showed enhanced pH stabilities than wild type, what is possible explanation for this.
At present manuscript we determinate the pH optima not pH stability of laccase variants. Indeed, mutant forms Met199Gly and His165Ala/Met199Gly functionally active in a wider pH range (from 6.5 to 9) than the SgfSLwt and other mutant forms (table 4). At present time, we investigate this phenomenon using site-direction mutagenesis. This work in progress and will be the topic of the next article.
- Does author studied decolorization of dyes without redox mediator ABTS for all mutants and wild type, if mutant exhibit decolorization without redox mediator, it will significant. Also, does any difference in requirement of the redox mediator concentrations in mutant laccases than wild type?
Unfortunately, mutants as well as SgfSLwt cannot decolorize the dyes without the mediator ABTS. The concentration of ABTS and proteins in experiments was always the same for the correct comparison of catalytical activity. We plan to investigate the dependence of decolorization activity of mutants from redox mediator concentrations in future.
Author Response File: Author Response.pdf
Reviewer 3 Report
In this study, Kolyadenko and coworkers studied the factors that influence the catalytic performance of the two domain laccase by optimizing the substrate binding pocket of SgfSL. They found that the Met199Gly/Arg240His is a prospective 2D laccase variant with the highest activity under alkaline conditions. I recommend its publication with minor revisions.
- why does the residual activity of His165Ala/Met199Gly in Table 3 exceed 100%?
Author Response
In this study, Kolyadenko and coworkers studied the factors that influence the catalytic performance of the two domain laccase by optimizing the substrate binding pocket of SgfSL. They found that the Met199Gly/Arg240His is a prospective 2D laccase variant with the highest activity under alkaline conditions. I recommend its publication with minor revisions.
- Why does the residual activity of His165Ala/Met199Gly in Table 3 exceed 100%?
The residual activity of His165Ala/Met199Gly in presence of sodium azide increases and does not decrease as than SgfSLwt and other mutants. The stimulating effect of NaN3 on activity was noticed for His165Ala (Gabdulkhakov et al., 2019, ref №18). In our previous article we showed this effect for His165Ala. In this work, for double mutant form His165Ala/Met199Gly, a similar effect after the adding of NaN3 was observed. We have noticed it again, and a detailed study of this effect will be given in the following works. To date, we don't know the exact mechanism of this effect and just wish to emphasize the reproducibility of the result. One of the hypotheses is the participation of sodium azide which is charged under acidic conditions in proton transfer. According to this hypothesis, when replacing His165Ala, the azide binds with polar amino acids (Ser 295, Gln 292, and others) in the T3 channel, and acts as a proton donor. Such an effect of sodium azide for some proteins has been shown (Cooper and Barry “Azide as a Probe of Proton Transfer Reactions in Photosynthetic Oxygen Evolution”, 2008 ).
The crystallographic study of NaN3 binding sites at laccase using crystals of His165Ala and His165Ala/Met199Gly is in progress in our group.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
L214-222: The phrase “copper content” should be omitted from the text and the title of the section as copper content was not measured in this experiment, but the absorbance change. You can discuss it below in the Discussion section but not in Results section.
L328-L329: please, avoid the term “copper dissociation rate of Cu1 ions from the T1 center” as it was absorption decrease measurements and there is no data on “copper dissociation rate of Cu1 ions from the T1 center”.
Please, carefully read the text for misspellings and grammar.
Author Response
Dear reviewer, thank you for your comments
L214-222: The phrase “copper content” should be omitted from the text and the title of the section as copper content was not measured in this experiment, but the absorbance change. You can discuss it below in the Discussion section but not in Results section.
The titles and text of sections 2.5 and 4.3.3 were changed appropriately with your recommendation (lines 215-224, 233-237, 524-525, 535, 541-542). Our suggestion about the reason for decreasing in absorbance intensity at 600 nm after the heating was added to Discussion section 3.2 (line 343-346).
L328-L329: please, avoid the term “copper dissociation rate of Cu1 ions from the T1 center” as it was absorption decrease measurements and there is no data on “copper dissociation rate of Cu1 ions from the T1 center”.
This part was rewritten appropriately with your recommendation (line 340-348). We have deleted the “copper dissociation rate of Cu1 ions from the T1 center” and left our assumption about the reason for the loss in the peak intensities:
"In our point of view, the decreasing of the intensities of the peaks during the heating is associated with dissociation of Cu1 ions from T1 centers of SgfSLwt and Met199Gly, and not with reduction".
Please, carefully read the text for misspellings and grammar.
Thank you for your comment, we have read the text again and tried to correct our mistakes.
