The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

Abstract

Proteases are implicated in several aspects of the physiology of microorganisms, as well as in host-pathogen interactions. Aminopeptidases are also emerging as novel drug targets in infectious agents. In this study, we have characterized an aminopeptidase from the spirochete Borrelia burgdorferi, the causative agent of Lyme disease. The aminopeptidolytic activity was identified in cell extracts from B. burgdorferi by using the substrate leucine-7-amido-4-methylcoumarin. A protein displaying this activity was purified from B. burgdorferi by a twostep chromatographic procedure, yielding a ϳ300-kDa homo-oligomeric enzyme formed by monomers of ϳ50 kDa. Gel enzymography experiments showed that enzymatic activity depends on the oligomeric structure of the protease but does not involve interchain disulfide bonds. The enzyme was identified by peptide mass fingerprinting as the putative aminopeptidase II of B. burgdorferi, encoded by the gene BB0069. It shares significant identity to members of the M29/T family of metallopeptidase, is sensitive to bestatin, has a neutral pH optimum, and displays maximal activity at 60°C. Its activity is 1.75-fold higher at the temperature of the mammalian host than at that of the insect host of the pathogen. The activity of this thermophilic aminopeptidase of B. burgdorferi (TAP Bb ) depends on Zn 2؉ , and temperatures over 70°C promoted its inactivation through a transition from the hexameric state to the monomeric state. Since B. burgdorferi is deficient in pathways for amino acid synthesis, TAP Bb could play a role in supplying required amino acids. Alternatively, the enzyme could be involved in peptide and/or protein processing.

The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases

“…Oligopeptidase B of T. cruzi also displays abnormal electrophoretic migration under the same experimental conditions [ 35 ]. Nevertheless, other enzymes such as T. cruzi cathepsin B and the hexameric leucyl aminopeptidase of Borrelia burgdorferi (TAP Bb ) show the expected migration [ 13 , 36 ]. The hexameric nature of LAPTc was thus confirmed by analytical ultracentrifugation and MALLS assays, which are accurate techniques to determine molecular masses of macromolecules in the absence of any interaction with matrices or surfaces.…”

Halotolerant aminopeptidase M29 from Mesorhizobium SEMIA 3007 with biotechnological potential and its impact on biofilm synthesis

“…At the same time, prolines represent only 5.4% and 4.1% of the residues in the amino acid sequences of Amp T and PePS, respectively. This finding suggests that other factors are responsible for their thermal stability such as the presence of disulfide bonds formed by cysteine residues that stabilize proteins by decreasing the entropy of the protein’s unfolded state 30 and/or oligomeric state formation, representing an adaptive advantage relative to their monomeric counterparts 24 . The ability to act at temperatures over 40 °C is of great biotechnological importance, reducing the risk of contamination by mesophilic microorganisms.…”

“…To date, in addition to the genes observed in bacteria, the ones encoding M29 aminopeptidases have been identified in animals, plants and Archea. However, a highly limited number of aminopeptidases M29 from Thermus aquaticus , Thermus thermophilus , Bacillus stearothermophilus , Streptococcus thermophilus , S. pneumoniae , S. aureus , Borrelia burgdorferi and Listeria monocitogenes have been characterized 13 , 20 , 24 , 25 . In this study, we present the results of genomic data mining in Mesorhizobium sp., as well as the cloning, overproduction in E. coli , and characterization of a highly halo-tolerant and solvent-tolerant aminopeptidase.…”

“… Name M.O. Characteristic Reference leucine aminopeptidase II Bacillus stearothermophilus - Homodimer- pH and temperature optimum for the hydrolysis reaction was pH 8.0 and 60 °C.- Co 2+ ions have a stimulatory effect 76 Aminopeptidase Borrelia burgdorferi - Homohexameric- pH and temperature optimum for the hydrolysis reaction was pH 7.5 and 60 °C.- Zn 2+ ions have a stimulatory effect 24 Aminopeptidase T Listeria monocytogenes - Homodimer- Strongly stimulated by Co 2+ - Virulence Factor for infection 13 Aminopeptidase PepS Streptococcus thermophiles - Aminopeptidase PepS probably plays a pleiotropic role through its involvement in growth via nitrogen nutrition 12 Alkaline Aminopeptidase Bacillus mycoides - Monomer- pH optimum for the hydrolysis reaction was 9.0.- Co 2+ ions have a stimulatory effect- After 30 min preincubation at 45 °C the aminopeptidase retained only 12% of activity. 77 leucine aminopeptidase LAP Staphylococcus aureus - High activity in the presence of Mn 2+ - pH optimum for the hydrolysis reaction was 8.5- broad substrate range that extends beyond leucine 41 Thermostable aminopeptidase Aquifex aeolicus - pH and temperature optimum for the hydrolysis reaction was pH 8.0 to 8.5 and 80 °C.- Highly resistant to organic solvents.…”

Autochthonous probiotic Lactobacillus sp. in the diet of bullfrog tadpoles Lithobates catesbeianus improves weight gain, feed conversion and gut microbiota

“…used in this study might have enhanced nutrient utilization of tadpoles based on analysis of digestive enzymes produced by the probiotic bacterium, such as leucine arylamidase, valine arylamidase and cystine arylamidase. These enzymes promote protein degradation by binding amino acid residues and polypeptide chains (Bertin et al., ; Gonzales & Robert‐Baudouy, ; Matsui, Fowler, & Walling, ). However, acid phosphatase and naphthol‐AS‐BI‐phosphohydrolase contribute to the dephosphorylation of several molecules, including nucleotides, alkaloids, proteins and organic phosphorus present in the lipid layer of the plasma membranes of plant cells (Aoyama, Silva, Miranda, & Ferreira, ; Peres, Machado, Chitarra, & Lima, ).…”