Alanine racemase (Alr; E.C. 5.1.1.1) is an enzyme that catalyzes the interconversion of L-alanine and D-alanine using a pyridoxal 5-phosphate (PLP) as a cofactor (Tassoni et al. 2017). It provides D-alanine for the synthesis of peptidoglycan of the bacterial cell wall, D-alanine is directly involved in cross-linking of adjacent peptidoglycan strands and also present in lipoteichoic acids of Gram-positive bacteria (Liu et al. 2018; Ray et al. 2018). There are two isoforms (non-homologous) of the alanine racemase genes (
Numerous inhibitors were identified as able to affect the activity of alanine racemase (Kim et al. 2003a; Kim et al. 2003b). Many of these inhibitors were structural analogs of alanine; they interact with the enzyme-bound PLP, covalently bound to some eukaryotic PLP-dependent enzymes and lead to cellular toxicity (Toney 2005). PLP-related off-target effects could be overcome by using enzyme inhibitors that are not substrate analogs. Structure-based approach and molecular modeling have been employed to discover novel alanine racemase inhibitors, which are devoid of affinity for the PLP and hence off-target effects (Lee et al. 2013; Azam and Jayaram 2018).
In this study, we identified and purified the alanine racemase from
Strains and plasmids used in this study.
Strains/Plasmids | Description | Source |
---|---|---|
Isolated from infected | This study | |
Used for cloning and propagation of plasmids | Novagen | |
Used for protein expression | Invitrogen | |
This study | ||
This study | ||
This study | ||
This study | ||
pMD19-T | Carries ampR gene; used for cloning PCR product with A at 3’ ends | Takara |
pET 22b (+) | Carries ampR gene; used for expressing | Novagen |
The evolutionary distances of the phylogenic tree were computed using the p-distance method and are reported in the units of the number of amino acid differences per site. The bootstrap consensus tree inferred from 500 replicates was used to represent the evolutionary history (Felsenstein 1985). The analysis involved 10 amino acid sequences from
SiAlr and different concentrations of hydroxylamine (0.1, 1, and 10 mM) were added to the reaction mixture without the substrate, dialyzed in phosphate buffered saline for 40 min, and its activity was determined without the addition of PLP. The effect of Dithiothreitol (DTT) on the activity of SiAlr was also determined by incubating the enzyme in different concentrations of DTT (1 and 3 mM) for 30 minutes and the relative activity was measured. To confirm SiAlr is a PLP-dependent enzyme, the purified Alr was treated with 10 mM hydroxylamine and dialyzed to obtain the apoenzyme. The apoenzyme was incubated in different concentrations of PLP (0.01, 0.04, and 0.06 mM) and its relative activity was measured.
The deduced amino acid sequence has 76%, 67%, 63%, and 47% similarities with alanine racemase of
Multiple sequence alignment of SiAlr with sequences of other 10 species Alr suggested that some regions are conserved in SiAlr, which includes PLP binding motif near the N-terminus (AVVKANAYGHG) and the two catalytic amino acid residues of the active center (Lys 40 and Tyr 274). The eight residues making up the entry-way to the active site (inner layer: Ala 174, Tyr 273, Tyr 282 and Tyr 366; middle layer: Asp 166, Arg 298, Arg 318 and Ile 364) (Fig. 2).
Various chemicals and metal ions were reported to inhibit the activity of alanine racemases. The results revealed that the enzyme activity was inhibited by most of the metal ions, but markedly inhibited by Ni2+, Co2+ Zn2+, and Fe2+ (Fig. 5).
Effect of Hydroxylamine, DTT and PLP on SiAlr Activity.
Chemical | Concentration (mM) | Relative activity (%) |
---|---|---|
None | 100 (0.7) | |
Hydroxylamine | 0.1 | 21 (1.2) |
1 | 11 (0.8) | |
10 | 9 (1.4) | |
DTT | 1 | 27 (3.1) |
3 | 2 (0.8) | |
PLP | 0.01 | 56 (2.4) |
0.04 | 83 (1.5) | |
0.06 | 96 (2.7) |
The data were presented as mean (SD) from three independent enzyme reactions
We examined the role of PLP in the activity of SiAlr by resolving the enzyme to Apo-enzyme by hydroxylamine treatment. The Apo-enzyme completely lost its activity after treatment with 10 mM hydroxylamine. Addition of 0.01, 0.04, and 0.06 mM of PLP make the enzyme regained up to 56%, 83 % and 96% of its activity, respectively. The result indicated that SiAlr is a PLP-dependent enzyme that requires more than 0.01 mM PLP to maintain its activity (Table II).
The results of antimicrobial activity of homogentisic acid and hydroquinone inhibitors against numerous isolates of Gram-positive and Gram-negative bacteria.
Organism | aMIC (μg/ml) | |
---|---|---|
Hydro-quinone | Homogentisic Acid | |
25 (2.3) | 200 (5.6) | |
130 (7.9) | 210 (8.4) | |
150 (8.7) | 180 (11.4) | |
210 (13.7) | 250 (14.1) | |
180 (11.5) | 210 (12.3) | |
0 | 0 |
MIC, average values with standard deviations
The optimal pH and temperature of SiAlr were 9.5 and 35°C, respectively, which were similar to alanine racemase from
Many studies have focused on alanine racemase to develop antibacterial drugs for multiple bacterial species (Scaletti et al. 2012; Shrestha et al. 2017). Although the MIC values of homogentisic acid and hydroquinone were higher than that of D-cycloserine, which is a cyclic analog of alanine and exerted its inhibitory effect through interaction with the enzyme-bound PLP cofactor (Batson et al. 2017), both homogentisic acid and hydroquinone are not structural analogs of Alr as such they are not interfering with other PLP dependent enzymes, but directly interact with the active sites of alanine racemase (Wang et al. 2017). According to the results of antimicrobial activity assay, the two inhibitors are capable of inhibiting both Gram-positive and Gram-negative bacteria with various efficacies, except
Several alanine racemases have been identified and characterized form the
Purification and characterization of Alr from both Gram-positive and Gram-negative bacteria is an essential step towards an in-depth understanding of enzyme divers features, design new broad-spectrum antibiotics, and site-directed mutagenesis studies to improve the enzyme catalysis and stability. Hydroquinone and homogentisic acid are promising inhibitors of Alr that are capable of inhibiting the growth of both Grampositive and Gram-negative bacteria. Future investigation will focus on finding the physiological role of Alr, exploring new novel antimicrobial agents against