Abstract
Unbranched heterocytous cyanobacteria produce a number of serine peptidases. We have characterized several peptidases in the cell-free extracts of a true-branched N2-fixing cyanobacterium, Westiellopsis ramosa sp. nov. Upon substrate-gel zymography of intact filaments and heterocytes, five peptidase bands were resolved, whereas in vegetative cells, a single band was discernible. No band was detected in \({\text{NO}}_{3}^{ - } /{\text{NH}}_{4}^{ + }\)-grown cultures suggesting that the peptidases were present under diazotrophic conditions with much of them confined to heterocytes. Using salt precipitation and chromatography, a caseinolytic peptidase, called Wrp49, was purified which also demonstrated fibrinolytic activity. In SDS-PAGE, the purified peptidase was resolved into 17 and 27 kDa fragments. The enzyme in its native state exhibited Mr ≈ 49 kDa, and digested gelatin in a substrate gel at a corresponding position. The enzyme showed amidolytic activity on a plasmin specific substrate, D-Val-Leu-Lys p-nitroanilide. Moreover, a trypsin specific substrate, N-benzoyl-DL-Arg p-nitroanilide was hydrolyzed at an apparent Km = 0.195 mM and Vmax = 5 × 10−7 M s−1. The enzyme was stable in a wide pH and temperature range. While Ca2+ stimulated the activity; phenylmethane sulfonyl fluoride, leupeptin, EDTA and chelants were inhibitory. The activity of the EDTA-inactivated enzyme was completely restored upon adding Ca2+, suggesting that both compounds competed with each other in modulating the enzyme activity. The enzyme showed similarities with a Ca2+ stimulated subtilisin-like serine peptidase of Anabaena variabilis ATCC 29413, but also presented several unique features of metallopeptidases, such as the chelant’s response. Moreover, the N-terminal sequence (MTVENLARTGVGPGWR) did not match with any of the known peptidases.
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Acknowledgment
The authors wish to thank the Head of the Department of Biological Science, Rani Durgavati University, Jabalpur, for providing lab facilities.
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SNB and PS designed the experiments. ND and PS performed the experiments.
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12298_2017_497_MOESM1_ESM.tif
Supplementary material Fig. S1 Photomicrographs of (a) NH4Cl supplemented culture showing no heterocytes, poor growth with retracted cytoplasmic content visible inside the cells and branching (b) Culture supplemented with NaNO3 showing better growth than (a) along with the presence of branching; no heterocytes are visible, and (c) culture without external supplementation of nitrogen showing the presence of heterocytes (arrows). Scale bar = 5 µm (TIFF 594 kb)
12298_2017_497_MOESM2_ESM.tif
Supplementary material Fig. S2 Demonstration of casein dissolution activity. (a) Sonicated cells and (b) culture filtrate equivalent to 10 and 20 µg protein in 30 µL, and (c) buffer were applied to dry filter paper discs (diameter, 5 mm) and placed on surface of casein plates prepared by hardening 1% casein in 20 mM Tris–HCl, pH 8.0 with 1.5% agar. After overnight incubation at 37 °C, plates were overlaid with 5% (w/v) TCA to observe clearing zones (TIFF 225 kb)
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Dubey, N., Singh, P. & Bagchi, S.N. A calcium-stimulated serine peptidase from a true-branching cyanobacterium, Westiellopsis ramosa sp. nov.. Physiol Mol Biol Plants 24, 261–273 (2018). https://doi.org/10.1007/s12298-017-0497-9
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DOI: https://doi.org/10.1007/s12298-017-0497-9