Biochemical characterization of a recombinant plant class III chitinase from the pitcher of the carnivorous plant Nepenthes alata

ABSTRACT The haloarchaeon Haloferax mediterranei has shown promise for the economical production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a desirable bioplastic. However, little is known at present about the genes involved in PHBV synthesis in the domain Archaea. In this study, we cloned the gene cluster (phaEC Hme) encoding a polyhydroxyalkanoate (PHA) synthase in H. mediterranei CGMCC 1.2087 via thermal asymmetric interlaced PCR. Western blotting revealed that the phaE Hme and phaC Hme genes were constitutively expressed, and both the PhaEHme and PhaCHme proteins were strongly bound to the PHBV granules. Interestingly, CGMCC 1.2087 could synthesize PHBV in either nutrient-limited medium (supplemented with 1% starch) or nutrient-rich medium, up to 24 or 18% (wt/wt) in shaking flasks. Knockout of the phaEC Hme genes in CGMCC 1.2087 led to a complete loss of PHBV synthesis, and only complementation with the phaEC Hme genes together (but not either one alone) could restore to this mutant the capability for PHBV accumulation. The known haloarchaeal PhaC subunits are much longer at their C termini than their bacterial counterparts, and the C-terminal extension of PhaCHme was proven to be indispensable for its function in vivo. Moreover, the mixture of purified PhaEHme/PhaCHme (1:1) showed significant activity of PHA synthase in vitro. Taken together, our results indicated that a novel member of the class III PHA synthases, composed of PhaCHme and PhaEHme, accounted for the PHBV synthesis in H. mediterranei.

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Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding

PROTOPLASMA ◽

10.1007/s00709-014-0684-2 ◽

2014 ◽

Vol 252 (1) ◽

pp. 335-343 ◽

Cited By ~ 4

Author(s):

Vesna Hadži-Tašković Šukalović ◽

Mirjana Vuletić ◽

Ksenija Marković ◽

Tijana Cvetić Antić ◽

Željko Vučinić

Keyword(s):

Cell Walls ◽

Biochemical Characterization ◽

Covalent Binding ◽

Root Cell ◽

Maize Root ◽

Enzyme Properties ◽

Class Iii ◽

Root Cell Walls

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Myo3A, One of Two Class III Myosin Genes Expressed in Vertebrate Retina, Is Localized to the Calycal Processes of Rod and Cone Photoreceptors and Is Expressed in the Sacculus

Molecular Biology of the Cell ◽

10.1091/mbc.e02-06-0317 ◽

2003 ◽

Vol 14 (3) ◽

pp. 1058-1073 ◽

Cited By ~ 40

Author(s):

Andréa C. Dosé ◽

David W. Hillman ◽

Cynthia Wong ◽

Lorraine Sohlberg ◽

Jennifer Lin-Jones ◽

...

Keyword(s):

Cellular Localization ◽

Biochemical Characterization ◽

Expression Patterns ◽

Tail Domain ◽

Class Iii ◽

Feature Characteristic ◽

Filament Bundles ◽

Myosin Motors ◽

Rod And Cone Photoreceptors

The striped bass has two retina-expressed class III myosin genes, each composed of a kinase, motor, and tail domain. We report the cloning, sequence analysis, and expression patterns of the long (Myo3A) and short (Myo3B) class III myosins, as well as cellular localization and biochemical characterization of the long isoform, Myo3A. Myo3A (209 kDa) is expressed in the retina, brain, testis, and sacculus, and Myo3B (155 kDa) is expressed in the retina, intestine, and testis. The tails of these two isoforms contain two highly conserved domains, 3THDI and 3THDII. Whereas Myo3B has three IQ motifs, Myo3A has nine IQ motifs, four in its neck and five in its tail domain. Myo3A localizes to actin filament bundles of photoreceptors and is concentrated in the calycal processes. An anti-Myo3A antibody decorates the actin cytoskeleton of rod inner/outer segments, and this labeling is reduced by the presence of ATP. The ATP-sensitive actin association is a feature characteristic of myosin motors. The numerous IQ motifs may play a structural or signaling role in the Myo3A, and its localization to calycal processes indicates that this myosin mediates a local function at this site in vertebrate photoreceptors.

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