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. 2019 Jan 29;10(2):101.
doi: 10.3390/genes10020101.

Characteristics of the First Protein Tyrosine Phosphatase With Phytase Activity From a Soil Metagenome

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Free PMC article

Characteristics of the First Protein Tyrosine Phosphatase With Phytase Activity From a Soil Metagenome

Genis Andrés Castillo Villamizar et al. Genes (Basel). .
Free PMC article

Abstract

Protein tyrosine phosphatases (PTPs) fulfil multiple key regulatory functions. Within the group of PTPs, the atypical lipid phosphatases (ALPs) are known for their role as virulence factors associated with human pathogens. Another group of PTPs, which is capable of using inositol-hexakisphosphate (InsP₆) as substrate, are known as phytases. Phytases play major roles in the environmental phosphorus cycle, biotechnology, and pathogenesis. So far, all functionally characterized PTPs, including ALPs and PTP-phytases, have been derived exclusively from isolated microorganisms. In this study, screening of a soil-derived metagenomic library resulted in identification of a gene (pho16B), encoding a PTP, which shares structural characteristics with the ALPs. In addition, the characterization of the gene product (Pho16B) revealed the capability of the protein to use InsP₆ as substrate, and the potential of soil as a source of phytases with so far unknown characteristics. Thus, Pho16B represents the first functional environmentally derived PTP-phytase. The enzyme has a molecular mass of 38 kDa. The enzyme is promiscuous, showing highest activity and affinity toward naphthyl phosphate (Km 0.966 mM). Pho16B contains the HCXXGKDR[TA]G submotif of PTP-ALPs, and it is structurally related to PtpB of Mycobacterium tuberculosis. This study demonstrates the presence and functionality of an environmental gene codifying a PTP-phytase homologous to enzymes closely associated to bacterial pathogenicity.

Keywords: metagenomic library; metagenomics; phosphatases; phytases; promiscuous enzymes.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Strand, location, and BLAST results for all identified open reading frames (ORFs) of the pLP16 insert. * Partial ORFs.
Figure 2
Figure 2
Domain organization and alignment of the subloop in Pho16B (A). Architecture of Pho16B showing the positions of the protein tyrosine phosphatase (PTP) domain and the signal peptide. (B) Alignment and position of the P-loop motif (HCXXGKDRTG) in Pho16B and other related atypical lipid phosphatase (ALP) proteins. Pho16B (this study), UniProtKB codes: P96830 (Mycobacterium tuberculosis), A0PWE5 (Mycobacterium ulcerans), A1TVR0 (Acidovorax citrulli), Q5FI40 (Lactobacillus acidophilus), A1SQF4 (Nocardioides sp.), A3WD47 (Erythrobacter sp.), A1JIE8 (Yersinia enterocolitica), and Q2UMD6 (Aspergillus oryzae).
Figure 3
Figure 3
Neighbor-joining phylogenetic tree showing the position of Pho16B. The numbers at the nodes indicate levels of bootstrap support (range from 0 to 100) and were based on 500 replicates. Names correspond to the groups (G) described by Beresford et al. (2010) and their corresponding UniProtKB codes. Pho16B, this study; WP_042381880 (closest related PTP phosphatase from Streptacidiphilus melanogenes); P96839 (MptpB Mycobacterium tuberculosis); G1: A0QNM9 (Mycobacterium smegmatis), A0PWE5 (Mycobacterium ulcerans), A0A0H2ZU00 (Mycobacterium avium); G2: A1TVR0 (Acidovorax avenae), A1WCM2 (Acidovorax sp.), A5EJL6 (Bradyrhizobium sp.); G3: A5VME4 (Lactobacillus reuteri), A8YWZ0 (Lactobacillus helveticus), Q03Q47 (Lactobacillus brevis); G4: A8LX04 (Salinispora arenicola), Q0SFJ4 (Rhodococcus jostii), Q2G3Q6 (Novosphingobium aromaticivorans); G5: Q1GTR8 (Sphingopyxis alaskensis), Q2NBB0 (Erythrobacter litoralis); G6: A1JIE8 (Yersinia enterocolitica), A6T6R5 (Klebsiella pneumoniae), A8G8B5 (Serratia proteamaculans); G7: A7EL60 (Sclerotinia sclerotiorum), Q0UXK4 (Phaeosphaeria nodorum), Q4WNE5 (Neosartorya fumigata); G8: A5E2J1 (Lodderomyces elongisporus), A7TQ59 (Vanderwaltozyma polyspora), Q6BSM6 (Debaryomyces hansenii); G9: A6R6W3 (Ajellomyces capsulatus), Q2UG77 (Aspergillus oryzae), A1DAV8 (Neosartorya fischeri).
Figure 4
Figure 4
Effect of temperature and pH on Pho16B activity. (A) Temperature profile of Pho16B enzymatic activity. (B) pH profile of Pho16B enzymatic activity. All measurements were performed in triplicate. Specific activity values are expressed as percentages of the highest relative activity: 8.78 and 4.3 U/mg, for A and B, respectively.
Figure 5
Figure 5
Substrate specificity of Pho16B. Relative activity of Pho16B was measured at 10 mM substrate concentration. All measurements were performed in triplicate and under optimal pH and temperature conditions for enzyme activity. Specific activity values are expressed as percentages of the highest relative activity (13.89 U/mg).
Figure 6
Figure 6
Effect of different concentrations of metal ions and inhibitors on the activity of Pho16B. All measurements were performed in triplicate and under optimal pH and temperature conditions for the enzyme. Specific activity values expressed as percentages of the control reactions (no additions) 8.2 U/mg for both concentrations.

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