Heterologous expression and functional characterization of a GH10 endoxylanase from Aspergillus fumigatus var. niveus with potential biotechnological application

The H+proton-translocating inorganic pyrophosphatase (H+-PPase) family is composed of two phylogenetically distinct types of enzymes: K+-dependent and K+-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H+-PPase from the thermophilic bacteriumCarboxydothermus hydrogenoformans. Both PPi-hydrolyzing and PPi-energized H+translocation activities of the recombinant enzyme inEscherichia coliinner membrane vesicles are strictly K+-dependent. Here we deduce the K+requirement of all available H+-PPase sequences based on the K+dependence ofC. hydrogenoformansH+-PPase in conjunction with phylogenetic analyses. Our data reveal that K+-independent H+-PPases possess conserved Lys and Thr that are absent in K+-dependent H+-PPases. We further demonstrate that a A460K substitution inC. hydrogenoformansH+-PPase is sufficient to confer K+independence to both PPihydrolysis and PPi-energized H+translocation. In contrast, a A463T mutation does not affect the K+dependence of H+-PPase.

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Functional Characterization of Homo- and Heteromeric Channel Kinases TRPM6 and TRPM7

The Journal of General Physiology ◽

10.1085/jgp.200609502 ◽

2006 ◽

Vol 127 (5) ◽

pp. 525-537 ◽

Cited By ~ 257

Author(s):

Mingjiang Li ◽

Jianmin Jiang ◽

Lixia Yue

Keyword(s):

Heterologous Expression ◽

Divalent Cation ◽

Functional Characterization ◽

Ph Sensitivity ◽

Physiological Processes ◽

Heteromeric Channel ◽

Unitary Conductance ◽

Cation Permeability

TRPM6 and TRPM7 are two known channel kinases that play important roles in various physiological processes, including Mg2+ homeostasis. Mutations in TRPM6 cause hereditary hypomagnesemia and secondary hypocalcemia (HSH). However, whether TRPM6 encodes functional channels is controversial. Here we demonstrate several signature features of TRPM6 that distinguish TRPM6 from TRPM7 and TRPM6/7 channels. We show that heterologous expression of TRPM6 but not the mutant TRPM6S141L produces functional channels with divalent cation permeability profile and pH sensitivity distinctive from those of TRPM7 channels and TRPM6/7 complexes. TRPM6 exhibits unique unitary conductance that is 2- and 1.5-fold bigger than that of TRPM7 and TRPM6/7. Moreover, micromolar levels of 2-aminoethoxydiphenyl borate (2-APB) maximally increase TRPM6 but significantly inhibit TRPM7 channel activities; whereas millimolar concentrations of 2-APB potentiate TRPM6/7 and TRPM7 channel activities. Furthermore, Mg2+ and Ca2+ entry through TRPM6 is enhanced three- to fourfold by 2-APB. Collectively, these results indicate that TRPM6 forms functional homomeric channels as well as heteromeric TRPM6/7 complexes. The unique characteristics of these three channel types, TRPM6, TRPM7, and TRPM6/7, suggest that they may play different roles in vivo.

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