Influence of Charge Distribution at the Active Site Surface on the Substrate Specificity of Human Neutrophil Protease 3 and Elastase

PTP1B is a cytosolic protein tyrosine phosphatase that is a regulator of the kinase activity of the insulin receptor; the two protein tyrosine phosphatases LAR and CD45 are receptor type phosphatases crucially important to cell function. LAR also is involved in regulation of the insulin receptor while CD45 is critical for T-cell activation. Although LAR and CD45 are both transmembrane phosphatases, these enzymes manifest their phosphatase activity through a catalytic cytosolic domain. We have utilized X-ray coordinates of related phosphatases (RPTPalpha and RPTPµ) and comparative protein modeling to obtain molecular models of the D1 catalytic domains of CD45 and LAR. The models were tested using established protocols and found to be comparable to low resolution X-ray structures. The structure obtained for LAR was compared with the recently reported X-ray structure. Both the CD45-D1 and LAR-D1 structures were then compared to and contrasted with PTP1B. The active site of pockets of the three enzymes were found to be very uniform in structure and charge distribution. Also, the gross surface topology around the active site was found to be somewhat similar for the 3 phosphatases. However, there were significant differences in surface topology, and, more importantly, large changes in surface charge distribution. The differences between the surface features of these enzymes provide an explanation for the selectivity of inhibition by a number of peptides.

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Redesigning the Substrate Specificity of an Enzyme by Cumulative Effects of the Mutations of Non-active Site Residues

Journal of Biological Chemistry ◽

10.1074/jbc.274.4.2344 ◽

1999 ◽

Vol 274 (4) ◽

pp. 2344-2349 ◽

Cited By ~ 125

Author(s):

Shinya Oue ◽

Akihiro Okamoto ◽

Takato Yano ◽

Hiroyuki Kagamiyama

Keyword(s):

Substrate Specificity ◽

Active Site ◽

Cumulative Effects ◽

Active Site Residues

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Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

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