Review Reports - Metagenomic Type IV Aminotransferases Active toward (<i>R</i>)-Methylbenzylamine

Round 1

Reviewer 1 Report

Statkevičius et al. identified and characterized three PLP-fold type IV ATs from a metagenomic library isolated from the Curonian Lagoon which were active not only towards D-amino acids and (R)-amines. In particular, they found that two of them exhibited high thermostability. The work has been conducted logically and the results were presented clearly. Thus, the manuscript is recommended for publication after minor revision. The points that need to be addressed include:

1) In Figure 4 and 5, to better understand the difference of active pocket between AT-4421 and AT-872, substrate or product should be docked into the structure model of proteins.

2) In Figure S3, it is very difficult to distinguish the of various systems only using different color, so it should be better to combine color and symbol. And I didn’t find the definition of ACF. And I don’t think it is a good method to determine the activity of ATs using the UV absorption of acetophenone.

3) Please revise the references carefully, such as ref 2 “(80-.)”, ref 8 “1937 1403542”, ref 12, ref 18-20, ref 23, ref 25, and ref 31.

Author Response

1) In Figure 4 and 5, to better understand the difference of active pocket between AT-4421 and AT-872, substrate or product should be docked into the structure model of proteins.

We took this suggestion and changed Fig.4 (b). It now shows AT-872 with a docked methylbenzylamine bound to PLP in the active site, thus making it easier to see the differences between AT-872 and AT of Haliscomenobacter hydrossis. The active sites of AT-872 and AT-4421 almost identical, hence, we think that a comparison of these enzymes with a docked substrate would be superfluous.

Fig. S3 was changed. ATs that showed no activity are now transparent so ATs of interest together with appropriate controls are easier to distinguish.

Please see the attachment for figure.

Acetophenone based methods are cheap, quick, easy to use (Slabu et al., 2017) and are widely used in similar works (Zhai et al., 2019, Jeon et al., 2022).

3) Please revise the references carefully, such as ref 2 “(80-.)”, ref 8 “1937 1403542”, ref 12, ref 18-20, ref 23, ref 25, and ref 31.

References were revised.

Author Response File: Author Response.pdf

Reviewer 2 Report

Authors have carried out in silico screening of metagenomic type IV aminotransfereses (ATs), and identified three of them active towards ( R )-methylbenzylamine. This is a well structured and rather complete study of identification and characterization of PLP-fold type IV ATs , resulting in the determination of the amino acids and various chiral amines for which they are actives.

The manuscript contributes with two bionew catalysts of relevant interest for the synthesis of chiral amines. Before to be accepted, it requires of several corrections, as follows:

Figures and Tables

They must be named following the citation order in the manuscript. Methods section appears after the results & discusión sections. Tables should be given as proper Tables and not as part of a Figure. All their data must be provided with their corresponding errors bars (Figures) or standard errors (Talbes). The text size of Figure legends and axis must be increased, the same for the figures of the axis:

Table S2 being the first one cited on page 2 L72, should be named as Table S1

Figure S2 B) is a Table, so it should be given as a separate Table (Table S2). Thus, on page 3 L104 instead of (Table S2)” it should be indicated “(Figure S2 and Table S2)”. The following numbers of tables and figures should be afterwards rearanged along the manuscript.

Figure 3- Captions of this figure should indicate the reaction conditions

Figure 3- AT-1132 is a less active enzyme than the other two, but from this figure it looks to have the oposite specificity for some amino acids than AT-872 and AT-4421. Could it be of intestest this results for futher studies of structurally similar enzymes looking for a more active one than this one but with this oposite specificity?

English- some parts of the text should be revised. On page 4 L 110, a new phrase should be started after ref 31

Results section

Page 4 L129- The phrase should be better indicating the temperatura of study: “At 30ºC, AT-4421 was moderately stable….”. Also, on L130 it should read: “…a significant decrease with time…”

Page 4, L134- The fact that AT-1132 showed an increase in activity after disolution and incubation at all the studied temperatures (≥30ºC) -not only at elevated ones- should be better described and justified by authors, as activities higher than 100% are maintained for longer time of incubation at lower temperatures…(Figure 2B)

Page 5 L151-This phrase should be better as follows: “…were the most active biocatalysts for pyruvated substrate,….”

Page 5 L152- Please specify which are the refered amino acceptor specificities

Page 5 L180. It should better reads: “These activity values towards R-MBA are similar to…”

Page 6 L185-186: it should better reads: “while AT-872 was even more active than this one with both, R-ATETR and ATETR analog. More precisely, AT-872 resulted ˃20% more active than AT-4421,…”

Materials & Methods.

Page 8 L263. Table S1 should be renamed

Page 8 L278, 281 and 300- please check the style of the journal and apply conveniently to the units of agitation speed. Is the use of an asterisk necessary?

In all the experiments, the number of replicas must be indicated, as well as, the system used for errors calculation

Conclusions

This section provides the essence of authors findinds, but the text needs improvement, for example, omiting some repeated issues and using an impersonal style. I suggest something like:

“Two PLP-fold type IVATs active towards D-amino acis and various transaminated ( R ) -amines have been identified and characterized…..” followed but the rest of authors text

Author Response

Figures and Tables

Table S2 being the first one cited on page 2 L72, should be named as Table S1

Table numbering was corrected.

Separate table was created.

Figure 3- Captions of this figure should indicate the reaction conditions

Fig. 3 caption now is: Substrate specificity of ATs. Pyruvate was used as amino acceptor for all reactions except those marked with “*” symbol where α-ketoglutarate was used. The detailed description of reaction conditions is outlined in the methods section. The reaction (100 μL) conditions: 5 mM of the amino donor, 5 mM of pyruvate or α-ketoglutarate, 10 μM PLP, 5 μg of the purified enzyme, and 50 mM sodium carbonate buffer (pH 9). The reactions were performed at 30 °C for 16 h.

AT-1132 is a branched chain amino acid aminotransferase, therefore, activity with L-amino acids is expected. AT-872 and AT-4421 are D-amino acid aminotransferases and are active towards D-amino acids. We agree that further analysis including site specific mutants would be very interesting, however that is out of scope of this study.

English- some parts of the text should be revised. On page 4 L 110, a new phrase should be started after ref 31

It now reads “In addition, ATs showed no activity with o-xylenediamine, a common chromogenic amino donor used for AT screening and activity measurements [31]. However, some of tested enzymes showed a low but measurable activity in the presence of another chromogenic amino donor, 4-nitrophenylethylamine [32] (Figure S3).”

Results section

Thermostability section was changed according to the suggestions: “The thermostability of the selected ATs was investigated at three temperatures (Figure 2b). At 30 °C, AT-4421 was moderately stable, the activity decreases by 30% after incubation for 24 hours. At higher temperatures, a significant decrease with time in AT-4421 activity was observed: 25% of activity was retained after 4 h at 50 °C, while at 70 °C complete inactivation occurred after 15 min. The other two enzymes were quite thermostable. AT-872 retained about 55% and AT-1132 around 80% of their activities even after 24 hours of incubation at 50 °C. Moreover, these two ATs retained more than 60% of their activity after incubation at 70 °C for 4 hours. AT-1132 showed an increase in activity after incubation at all tested temperatures. Such phenomena was observed with certain other thermostable ATs and might be explained by the refolding of a protein that was kept at -20°C to its natural conformation [34,35]. This increase in activity (above 100%) lasted longer at lower temperatures due to slower protein denaturation rates.”

Page 5 L151-This phrase should be better as follows: “…were the most active biocatalysts for pyruvated substrate,….”

Page 5 L152- Please specify which are the refered amino acceptor specificities

This section was changed to “We measured ATs’ preferences for different amino acceptors (Table 2). AT-872 and AT-4421 were the most active in the presence of pyruvate, while AT-1132 had the highest activity towards α-ketoglutarate.

The AT-1132 is closely related to BCATs according to protein sequence and its specificity for amino acceptor is similar to BCATs, which are most active with α-ketoglutarate. While AT-872 and AT-4421 are homologous to the DAATs, which prefer pyruvate as an amino acceptor [13,36,37]. Thus, amino acceptor preference of selected ATs corelates with protein sequences.”

Page 5 L180. It should better reads: “These activity values towards R-MBA are similar to…”

Page 6 L185-186: it should better reads: “while AT-872 was even more active than this one with both, R-ATETR and ATETR analog. More precisely, AT-872 resulted ˃20% more active than AT-4421,…”

This section was changed to: “AT-1132 was able to deaminate L- and D-alanine and some L-amino acids, although with a very low efficiency. AT-872 and AT-4421 were active with D-amino acids and L-alanine. None of the ATs tested were able to use β-alanine or isopropylamine (ISP) as an amino donor. All three ATs were active with amino donor R-MBA (AT-872 – 1.0 U/mg, AT-1132 – 0.2 U/mg, and AT-4421 – 0.9 U/mg). These activity values to-wards R-MBA are similar to that of the other described PLP-fold type IV ATs [25,29]. AT-872 and AT-4421 were also able to deaminate (R)-pyridylethylamine (R-PEA). Although a small amount of keto product was detected, the activity of AT-1132 with R-PEA was too low to give an accurate estimate of the conversion. Interestingly, AT-4421 was able to deaminate the bulky substrate (R)-aminotetralin (R-ATETR), while AT-872 was also active with the R-ATETR analog (R)-hydroxyaminotetralin (R-HATETR) and showed more than 20% conversion using equal amounts of amino donor and amino acceptor.”

Materials & Methods.

Page 8 L263. Table S1 should be renamed

Table S1 was renamed to Table S3.

Page 8 L278, 281 and 300- please check the style of the journal and apply conveniently to the units of agitation speed. Is the use of an asterisk necessary?

Unit were changed to 4,000× g; 15,000× g and 10,000× g.

In all the experiments, the number of replicas must be indicated, as well as, the system used for errors calculation

Where applicable number of replications are now stated in the methods section and error bars in the figures are now explained.

Conclusions

This section provides the essence of authors findinds, but the text needs improvement, for example, omiting some repeated issues and using an impersonal style. I suggest something like:

“Two PLP-fold type IVATs active towards D-amino acis and various transaminated ( R ) -amines have been identified and characterized…..” followed but the rest of authors text

Conclusions were changed to “Several PLP-fold type IV ATs were identified and characterized in this study. These enzymes are not only active towards D-amino acids, but can also transaminate various (R)-amines, which makes them potential biocatalysts. Based on the high thermostability of AT-872 and AT-1132, and the broad substrate spectrum of AT-872 and AT-4421, the engineering of these ATs for application in the production of high-value amino compounds seems feasible. Our results suggest that BCAT and DAAT have bio-catalytic potential and that these groups of fold type IV aminotransferases should not be overlooked in the search for R-selective ATs.”

Author Response File: Author Response.pdf