Kinetic and Structural Properties of a Robust Bacterial L-Amino Acid Oxidase

Round 1

Reviewer 1 Report

The authors carry out the analysis of the kinetic and structural properties of a marine bacterial L-amino acid oxidase, by producing it efficiently in E. coli, purifying it for biochemical analysis and further purification for crystallography. They determine good working conditions in terms of pH, temperature, and salt concentration for the enzyme, and then assay its activity on the twenty L-amino acids found in proteins. This enzyme prefers leucine, methionine, phenylalanine and glutamine. The reasons for these preferences are borne out in the crystallographic structure of the binding pocket for the substrate abutting the FAD system of rings that accepts the electron pair from the amino group of the amino acid, converting it first into an imino group and later into a keto group.

 

I consider that this is a well-written article, describing careful work, and suggest only minor revisions as follows:

• regarding expression and purification, I want to know where the His-tag is located as this could impact dimer association, and whether the protein was directed to the periplasmic space (this would explain the low toxicity to the host).
• the analysis of the favorite pH, salt concentration, and temperature lacks plots to show the trends. These could be another set of supplementary figures. Given that the enzyme is native to a marine bacterium, the activity increase at 500 mM NaCl is not surprising.
• I would welcome a small discussion on why this enzyme should be thermostable. Is its original host subject to extreme temperatures?
• regarding the kinetics of the enzyme measured for the twenty amino acids, an explanation to why this is done at 37 Celsius and not at the optimal working temperature of the enzyme would be welcome. Ditto for the salt concentration. I understand that activity is not measured directly, but by the consumption of hydrogen peroxide, and that could limit both temperature and salt concentrations, but this should be stated, not left to the imagination.
• regarding the structure, again the issue of the salt concentration comes to light. I understand that obtaining good crystals depends on multiple variables, including luck, but I would like to know if high salt (0.5 M NaCl) was even considered. This would be important for checking the importance of the salt bridges between the subunits.
• about the entrance to the active site, I think that various orientations of Supplementary Figure 7 would be useful. From looking at the structure of the ancestral LAAO and one of the viper ones, there indeed seems to be a constriction near the FAD triple ring, which would imply motion of the sidechains to allow entry to the active site.

Author Response

regarding expression and purification, I want to know where the His-tag is located as this could impact dimer association, and whether the protein was directed to the periplasmic space (this would explain the low toxicity to the host).

• The Pl-LAAO was expressed with an N-terminal His6-tag. For clarity, the complete protein sequence is now provided in the supplementary information. Also, the type of expression vector has been added (pBAD). The protein was purified from cells, confirming their intracellular localisation as recombinantly expressed protein.

the analysis of the favorite pH, salt concentration, and temperature lacks plots to show the trends. These could be another set of supplementary figures. Given that the enzyme is native to a marine bacterium, the activity increase at 500 mM NaCl is not surprising.

• Plots of effects of salt, pH and temperature on Pl-LAAO stability and activity have been added to the supplementary information (Fig. S4).

I would welcome a small discussion on why this enzyme should be thermostable. Is its original host subject to extreme temperatures?

• We have added a hypothesis on the reason why Pl-LAAO is relatively robust in the Discussion: “The relatively high temperature tolerance and preference for high salt concentrations may reflect the conditions at which the oxidase is active in real life. It has been found that it is secreted by the original bacterial host, contributing to the antimicrobial activity of Pseudoalteromonas luteoviolacea [14].”

regarding the kinetics of the enzyme measured for the twenty amino acids, an explanation to why this is done at 37 Celsius and not at the optimal working temperature of the enzyme would be welcome. Ditto for the salt concentration. I understand that activity is not measured directly, but by the consumption of hydrogen peroxide, and that could limit both temperature and salt concentrations, but this should be stated, not left to the imagination.

• We have chosen the same conditions as used in the previous study (ref #14) on this enzyme to allow comparison of both studies.

regarding the structure, again the issue of the salt concentration comes to light. I understand that obtaining good crystals depends on multiple variables, including luck, but I would like to know if high salt (0.5 M NaCl) was even considered. This would be important for checking the importance of the salt bridges between the subunits.

• We have used various standard crystallisation screening conditions which resulted in well diffracting crystals using PEG. As these crystals could be used to elucidate the structure at high resolution, we did not pursue other conditions.

about the entrance to the active site, I think that various orientations of Supplementary Figure 7 would be useful. From looking at the structure of the ancestral LAAO and one of the viper ones, there indeed seems to be a constriction near the FAD triple ring, which would imply motion of the sidechains to allow entry to the active site.

• As requested, we have added two figures (panels) to Figure S7. Still, for a good view on the structural features of this and other amine oxidases, for which the structures are available at the PDB database.

Reviewer 2 Report

In the present manuscript, the authors describe the kinetic and structural characterization of a L-amino acid oxidase from bacterial source, which was heterologously expressed in a previous published work (Andreo-Vidal, A.; Sanchez-Amat, A.; Campillo-Brocal, J.C. The Pseudoalteromonas luteoviolacea L-amino acid oxidase with anti-320 microbial activity is a flavoenzyme. Mar Drugs. 2018, 16, 499.). The text is well written, and the experiments are well organized and properly conducted. In my opinion, the manuscript could be published with a few minor revisions, as follows:

 

Introduction, L 45-46: Please provide references for the mentioned biotechnological applications.

Results, L65: Abbreviations must be explained at the first mention

L79-97: Please include a figure to better illustrate these results, which could also be included in the Supplementary material section.

Table 1: Please include the Vmax (U/mg) in the table.

Materials and methods: L215-217: Please provide the composition of TB medium. Was FAD supplemented in the culture medium?

Author Response

We thank the reviewer for the positive and constructive comments. We used the comments to improve the quality of the manuscript. Details are below:

- Introduction, L 45-46: Please provide references for the mentioned biotechnological applications.

> References were added, the others have been properly renumbered.

- Results, L65: Abbreviations must be explained at the first mention

> Abbreviations have been added.

- L79-97: Please include a figure to better illustrate these results, which could also be included in the Supplementary material section.

A figure (Figure S4 in the SI) showing the data for thermostability has been added.

- Table 1: Please include the Vmax (U/mg) in the table.

As request, the values have been included in the table.

- Materials and methods: L215-217: Please provide the composition of TB medium. Was FAD supplemented in the culture medium?

A description of the composition of TB medium has been added. FAD was not added to the culture medium.

Reviewer 3 Report

In this work, Simone Savino and colleagues characterize an amine oxidase from the bacterium Pseudoalteromonas luteoviolacea. They determine the kinetic parameters towards the proteinogenic L-aminoacids, the thermostability, and the crystal structure of this enzyme.
I fully agree with the authors that this study provides a solid basis for the development of engineered LAAO variants to be used in biocatalytic processes
The abstract is set out well. The background information that leads to the work is explained well and the introduction is appropriate with all the relevant literature references. The manuscript is written well, and the findings are sound.
Minor points: one of the most striking aspects of this work is the high level of expression of the recombinant protein. Certainly, the optimization on the codon usage of E. coli helps, but an important contribution is given by the plasmid used. As the authors do not clearly indicate this in “materials and methods” I suggest adding this data.

I also suggest making the supporting info legends more descriptive. in particular, in figure s4 legend the authors should indicate the number of replicas performed considering that error bars are reported in all the graphs.

Author Response

We thank the reviewer for the positive and constructive comments which we have used to improve the quality of the manuscript. Details are below:

> we have now mentioned the expression vector for expression of the enzyme (pBAD).

> We have improved several legends in the supplementary information. Also, as requested, it is now indicated that the kinetic data were measured in duplicate.