Influence of tryptic hydrolysis on the enzymatic function of the membrane-bound form of particulate methane monooxygenase from Methylosinus trichosporium OB3b.

Abstract:

:Particulate methane monooxygenase (pMMO) is a membrane protein embedded in the intracytoplasmic membrane of methane-oxidizing bacteria. Structural analysis of pMMO showed the existence of a hydrophilic region exposed outside of the bacterial membrane. To obtain information regarding the role of this hydrophilic region in the enzymatic function of pMMO, trypsin proteolysis of the membrane-bound form of pMMO from Methylosinus trichosporium OB3b was performed at 4 °C. The polypeptides produced by this hydrolysis were analyzed by polyacrylamide gel electrophoresis and MALDI-TOF/TOF. Furthermore, the influence of this tryptic digestion on the methane hydroxylation and propene epoxidation enzymatic activities of pMMO was investigated. Among the three subunits of pMMO, PmoB and PmoC were hydrolyzed by trypsin, but PmoA was not. With 10 mg L-1 trypsin, both terminal regions or the C-terminal region of PmoC polypeptide was selectively hydrolyzed. Furthermore, the stability of pMMO was decreased by this digestion. These results indicate that PmoC plays a role in maintaining the stability of pMMO in vitro. On the other hand, the digestion of PmoB with 100 mg L-1 trypsin produced several polypeptides, indicating that trypsin digestion occurs at several sites of the hydrophilic region of PmoB. Hydrolysis led to a decrease in pMMO activity towards methane hydroxylation and propene epoxidation. These results indicate that the hydrophilic region of PmoB is critically important for the enzymatic function of pMMO, which is consistent with the models of the functional mechanism of pMMO proposed so far.

journal_name

J Biotechnol

journal_title

Journal of biotechnology

authors

Miyaji A,Satou K,Baba T

doi

10.1016/j.jbiotec.2020.08.006

subject

Has Abstract

pub_date

2020-11-10 00:00:00

pages

98-106

eissn

0168-1656

issn

1873-4863

pii

S0168-1656(20)30219-4

journal_volume

323

pub_type

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