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

Abstract

Streptococcus iniae is a pathogenic and zoonotic bacteria that impacted high mortality to many fish species as well as capable of causing serious disease to humans. Alanine racemase (Alr, EC 5.1.1.1) is a pyridoxal-5’-phosphate (PLP)-containing homodimeric enzyme that catalyzes the racemization of L-alanine and D-alanine. In this study, we purified alanine racemase from S. iniae that was isolated from an infected Chinese sturgeon (Acipenser sinensis), as well as determined its biochemical characteristics and inhibitors. The alr gene has an open reading frame (ORF) of 1107 bp, encoding a protein of 369 amino acids, which has a molecular mass of 40 kDa. The enzyme has optimal activity at a temperature of 35°C and a pH of 9.5. It belongs to the PLP-dependent enzymes family and is highly specific to L-alanine. S.iniaeAlr (SiAlr) could be inhibited by some metal ions, hydroxylamine and dithiothreitol (DTT). The kinetic parameters Km and Vmax of the enzyme were 33.11 mM, 2426 units/mg for L-alanine, and 14.36 mM, 963.6 units/mg for D-alanine. Finally, the 50% inhibitory concentrations (IC50) values and antibiotic activity of two alanine racemase inhibitors (homogentisic acid and hydroquinone), were determined and found to be effective against both Gram-positive and Gram-negative bacteria employed in this study.

NMR-Metabolomic Profiling and Genome Mining Drive the Discovery of Cyclic Decapeptides from a Marine Streptomyces

“…Ala 1 was assigned as d -Ala according to Marfey’s analysis (Figures S23 and S24), which is proposed to be generated through racemization of l -Ala by the alanine racemase (Alr; E.C. 5.1.1.1; Figure S26), a class of conserved bacterial enzymes belonging to the type III pyridoxal phosphate (PLP)-dependent enzymes . Unexpectedly, in module 2, assembly is carried out between d -Ala 1 and N Me- l -Phe 2 under catalysis of the L C L domain.…”

“…5.1.1.1; Figure S26), a class of conserved bacterial enzymes belonging to the type III pyridoxal phosphate (PLP)-dependent enzymes. 30 Unexpectedly, in module 2, assembly is carried out between D-Ala 1 and NMe-L-Phe 2 under catalysis of the L C L domain. This mechanism needs further study.…”

Proteomics based systematic exploration of the peptidoglycan biosynthesis of Olsenella uli DSM 7084 towards pathogenesis

“…Alanine racemase is considered an excellent antibacterial drug target in several gram-positive pathogens, for example, Streptococcus sp. [39], Klebsiella sp. [40], Mycobacterium sp.…”

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“…The probable target sites [10][11][12][13]8 in cell wall synthesis are summarized in Fig. 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] .…”

“…Sources: Aebutus unedo (strawberry-tree), honey, Xanthomonas campestris pv. Phaseoll, Yarrownia lipolytica Alr-2 inhibitors found to be active against Aeromonas hydrophila IC 50 value= 0.2 µM MIC value= 1.73 µg/mL Potent inhibitor of Alr isolated from Streptococcus iniae HNM-1 18,33,34 9.…”