A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3

Abstract

We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea.

Purification, Characterization and Inhibition of Alanine Racemase from a Pathogenic Strain of Streptococcus iniae

“…SiAlr requires exogenous PLP for its activity in contrast to Alr from B. pseudofirmus OF4 and Thalassiosira sp., which does not require exogenous PLP for optimal activity (Ju et al 2009). The enzyme showed high substrate specificity to alanine, which is similar to most of the characterized alanine racemases (Kawakami et al 2018). Many studies have focused on alanine racemase to develop antibacterial drugs for multiple bacterial species (Scaletti et al 2012;Shrestha et al 2017).…”

“…The bacterial cell wall is indispensable for the survival and viability of bacteria (Liu et al 2019) and has always been an attractive target for many antibiotics and antimicrobial agents (Anthony et al 2011). Alanine racemase is ubiquitous among bacteria and rare in eukaryotes but absent in humans (Kawakami et al 2018); hence, it emerges as an attractive and potential therapeutic target for the antimicrobial drugs development (Wang et al 2017).…”

Cloning, Biochemical Characterization and Inhibition of Alanine racemase fromStreptococcus iniae

“…SiAlr is a mesophilic enzyme, stable at a temperature of 0 0 C to 40 0 C. Thermal stability of an enzyme is correlated with the host bacteria physiology and environment, thermophilic bacteria Alr are more stable than mesophilic and psychrotroph bacteria (Soda and Tanizawa, 1990;Yokoigawa et al, 1993 enhanced racemization of alanine racemase (Liu et al, 2015). SiAlr was characterized as PLP-dependent racemase and showed high substrate specificity to alanine which is similar to most of the characterized Alanine racemases (Kawakami et al, 2018).…”

“…The bacterial cell wall is indispensable for the survival and viability of bacteria and has always been an interesting target for many antibiotics and antimicrobial agents (Anthony et al, 2011). Alanine racemase is ubiquitous among bacteria and rare in eukaryotes but absent in humans (Kawakami et al, 2018), hence it emerges as an attractive and potential therapeutic target for the antimicrobial drug (Wang et al, 2017). Numerous inhibitors were identified as able to affect the activity of alanine racemase (Kim et al, 2003a;Kim et al, 2003b).…”

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“…2 This bacterial enzyme execute predominating role in cell wall synthesis of bacteria 14, 15 by providing D-alanine (D-ala) which serve as a key molecule for the biosynthesis of peptidoglycan network of mycobacterial cell wall; hence its inhibition has been reported to be fatal to pathogen viability in the deprivation of D-alanine supplementation 16,17 . The lipid-rich mycobacterial cell wall is common amidst prokaryotes, making Alanine racemase a putative target for the design and development of pharmacologically active drugs [18][19][20][21] . D-alanine provided by Alanine racemase is utterly vital for maintaining the growth and integrity of the cell wall.…”