Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC–MS/MS based protein quantification and metabolic flux analysis

Abstract

Flowering plants have syringyl and guaiacyl subunits in lignin in contrast to the guaiacyl lignin in gymnosperms. The biosynthesis of syringyl subunits is initiated by coniferaldehyde 5-hydroxylase (CAld5H). In Populus trichocarpa there are two closely related CAld5H enzymes (PtrCAld5H1 and PtrCAld5H2) associated with lignin biosynthesis during wood formation. We used yeast recombinant PtrCAld5H1 and PtrCAld5H2 proteins to carry out Michaelis–Menten and inhibition kinetics with LC–MS/MS based absolute protein quantification. CAld5H, a monooxygenase, requires a cytochrome P450 reductase (CPR) as an electron donor. We cloned and expressed three P. trichocarpa CPRs in yeast and show that all are active with both CAld5Hs. The kinetic analysis shows both CAld5Hs have essentially the same biochemical functions. When both CAld5Hs are coexpressed in the same yeast membranes, the resulting enzyme activities are additive, suggesting functional redundancy and independence of these two enzymes. Simulated reaction flux based on Michaelis–Menten kinetics and inhibition kinetics confirmed the redundancy and independence. Subcellular localization of both CAld5Hs as sGFP fusion proteins expressed in P. trichocarpa differentiating xylem protoplasts indicate that they are endoplasmic reticulum resident proteins. These results imply that during wood formation, 5-hydroxylation in monolignol biosynthesis of P. trichocarpa requires the combined metabolic flux of these two CAld5Hs to maintain adequate biosynthesis of syringyl lignin. The combination of genetic analysis, absolute protein quantitation-based enzyme kinetics, homologous CPR specificity, SNP characterization, and ER localization provides a more rigorous basis for a comprehensive systems understanding of 5-hydroxylation in lignin biosynthesis.

DOI: 10.1007/s00425-012-1663-5

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@article{Wang2012FunctionalRO, title={Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC–MS/MS based protein quantification and metabolic flux analysis}, author={Jack Peng-Yu Wang and Christopher M Shuford and Quanzi Li and Jina Song and Ying-Chung Jimmy Lin and Ying-Hsuan Sun and Hsi-Chuan Chen and Cranos M. Williams and David C Muddiman and Ronald R. Sederoff and Vincent Chiang}, journal={Planta}, year={2012}, volume={236}, pages={795-808} }