Class I Polyhydroxyalkanoate Synthase from the Purple Photosynthetic Bacterium Rhodovulum sulfidophilum Predominantly Exists as a Functional Dimer in the Absence of a Substrate

ACS Omega
Mieko Higuchi-TakeuchiKeiji Numata

Abstract

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters that accumulate as carbon and energy storage compounds in a variety of micro-organisms. The marine purple photosynthetic bacterium Rhodovulum sulfidophilum is capable of synthesizing PHA. In this study, we cloned a gene encoding a class I PHA synthase from R. sulfidophilum (phaC Rs ) and synthesized PhaCRs using a cell-free protein expression system. The specific activity of PhaCRs increased linearly as the (R)-3-hydroxybutyryl-coenzyme A (3HB-CoA) concentration increased and never reached a plateau, even at 3.75 mM 3HB-CoA, suggesting that PhaCRs was not saturated because of low substrate affinity. Size exclusion chromatography and native polyacrylamide gel electrophoresis analyses revealed that PhaCRs exists predominantly as an active dimer even in the absence of 3HB-CoA, unlike previously characterized PhaCs. The linear relationship between the PhaCRs activity and 3HB-CoA concentrations could result from a low substrate affinity as well as the absence of a rate-limiting step during PHA polymerization because of the existence of predominantly active dimers.

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Citations

Jun 14, 2019·Frontiers in Bioengineering and Biotechnology·Mieko Higuchi-Takeuchi, Keiji Numata
Nov 5, 2019·Frontiers in Bioengineering and Biotechnology·Mieko Higuchi-Takeuchi, Keiji Numata

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Datasets Mentioned

BETA
ADP70651
EPY03196

Methods Mentioned

BETA
electrophoresis
size exclusion chromatography
PCR
protein assay
Assay

Software Mentioned

BPROM
MUSCLE

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