Structural Determinants of the Specific Activities of an L-Amino Acid Oxidase from Pseudoalteromonas luteoviolacea CPMOR-1 with Broad Substrate Specificity

Abstract

The Pseudoalteromonas luteoviolacea strain CPMOR-1 expresses a flavin adenine dinucleotide (FAD)-dependent L-amino acid oxidase (LAAO) with broad substrate specificity. Steady-state kinetic analysis of its reactivity towards the 20 proteinogenic amino acids showed some activity to all except proline. The relative specific activity for amino acid substrates was not correlated only with K_m or k_cat values, since the two parameters often varied independently of each other. Variation in K_m was attributed to the differential binding affinity. Variation in k_cat was attributed to differential positioning of the bound substrate relative to FAD that decreased the reaction rate. A structural model of this LAAO was compared with structures of other FAD-dependent LAAOs that have different substrate specificities: an LAAO from snake venom that prefers aromatic amino acid substrates and a fungal LAAO that is specific for lysine. While the amino acid sequences of these LAAOs are not very similar, their overall structures are comparable. The differential activity towards specific amino acids was correlated with specific residues in the active sites of these LAAOs. Residues in the active site that interact with the amino and carboxyl groups attached to the α-carbon of the substrate amino acid are conserved in all of the LAAOs. Residues that interact with the side chains of the amino acid substrates show variation. This provides insight into the structural determinants of the LAAOs that dictate their different substrate preferences. These results are of interest for harnessing these enzymes for possible applications in biotechnology, such as deracemization.

Keywords: enzyme; flavoprotein; hydrogen peroxide; kinetics; phylogenetic relationship; protein structure–function.

Figure 1

Absorbance spectra of P. luteoviolacea CPMOR-1 LAAO. (A). Spectrum of the as-isolated protein in the oxidized state. (B). Spectrum immediately after the addition of 1 mM methionine. (C). Spectra after the addition of a 20-fold excess of sodium dithionite. The dashed line spectrum was recorded immediately after the addition and the solid line spectrum was recorded approximately 8 min after mixing. Spectra were recorded of 30 μM LAAO in 50 mM potassium phosphate, pH 7.0.

Figure 2

Structure model of P. luteoviolacea CPMOR-1 LAAO. The overall structure is shown as a cartoon on the left. The pink color indicates the regions in which differences in the amino acids between this enzyme and P. luteoviolacea CPMOR-2 LAAO were primarily located. FAD is shown with the carbons of the isoalloxazine and adenine rings yellow, nitrogens blue, oxygens red, and phosphorous orange. On the right are shown the residues in the substrate-binding area of the protein, which bind the amino acid substrates and position them for reaction with the FAD cofactor. Carbons of the amino acids are green.

Figure 3

Steady-state kinetic analysis of the reactions of select amino acid substrates with P. luteoviolacea CPMOR-1 LAAO. The analysis of the data is shown for (A) Leucine, (B) Methionine, (C) Glutamine, (D) Isoleucine, (E) Valine and (F) Glutamic acid. The lines are the fits of the data to Equation (1). Data points are the average of three replicates. The structures of each amino acid are shown with each plot.

Figure 4

Steady-state kinetic analysis of the reactions of aromatic amino acid substrates with P. luteoviolacea CPMOR-1 LAAO. The analysis of the data is shown for (A) Phenylalanine, (B) Tryptophan and (C) Tyrosine. The lines are the fits of the data to Equation (1). Data points are the average of three replicates. The structures of each amino acid are shown with each plot.

Figure 5

Amino acid sequence alignment of LAAOs. The sequences displayed are P. luteoviolacea CPMOR-1 LAAO (Pl LAAO1) (GenBank: WP_063369592), P. luteoviolacea CPMOR-2 LAAO (Pl LAAO2) (GenBank: KZN49687), SvLAAO (GenBank: CAB71136.), and TvLAAO (UniProt A0A0J9X1X3). Residues in the active site are highlighted. These are residues that interact with the carboxyl group (red) and amino group (cyan) bound to the α-carbon, residues that potentially interact with the amino acid side chain (green), and a residue that stabilizes a water molecule for use in catalysis (yellow). The numbering in this table corresponds to the amino acid numbers in the figures in this paper. It should be noted that the first 18 residues in the SvLAAO sequence comprise a signal sequence that is cleaved during export of the protein outside of the cell. The sequence in PDB 2IID does not include these residues. Therefore, there is an 18-residue difference in the numbering adopted here compared to the numbering in the PDB sequence.

Figure 6

Monomeric structures of LAAOs. (A) P. luteoviolacea CPMOR-1 LAAO. (B) SvLAAO from C. rhodostoma with Phe substrate bound (PDB 2IID) [28]. (C) TvLAAO (PBD 7C3H) [29] with Lys substrate bound. FAD is shown with the carbons of the isoalloxazine and adenine rings yellow, nitrogens blue, oxygens red, and phosphorous orange. The carbons of the Phe and Lys substrates are colored green. The additional helices present in P. luteoviolacea CPMOR-1 LAAO (A) that are not in the other LAAOs are colored red.

Figure 7

Active site residues that interact with the carboxyl and amino groups of the α-carbon of the amino acid substrate. (A) P. luteoviolacea CPMOR-1 LAAO. (B) SvLAAO from C. rhodostoma (PDB 2IID) with a Phe substrate present. The red sphere is a water molecule, which is coordinated by Lys344. (C) TvLAAO (PBD 7C3H) with a Lys substrate present. Oxygens are red, nitrogens are blue, and carbons are grey in the protein amino acids, yellow in FAD and green in the amino acid substrates.

Figure 8

Active site residues that interact with the side chains of the amino acid substrate. In contrast to Figure 7, the residues in the active site pocket that potentially interact with the substrate side chain are shown. (A) P. luteoviolacea CPMOR-1 LAAO. (B) SvLAAO from C. rhodostoma (PDB 2IID) with a Phe substrate present. (C) TvLAAO (PBD 7C3H) with a Lys substrate present. Oxygens are red, nitrogens are blue, and carbons are grey in the protein amino acids, yellow in FAD and green in the amino acid substrates.

Figure 9

Phylogenetic relationship of proteins with similarity to PlLAAO. The tree shown was constructed using the neighbor-joining method integrated in the program MEGA X [32]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Asterisk at the branches indicate bootstrap values higher than 90% for the phylogenetic analyses performed with both the neighbor-joining and maximum likelihood methods. Colored blue are the P. luteoviolacea strains CPMOR-1 and CPMOR-2 and the proteins from the non-bacterial species.