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Phe317 is essential for rubber oxygenase RoxA activity.

Jakob Birke¹, Nadja Hambsch, Georg Schmitt

¹Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany.

Applied and Environmental Microbiology

|September 4, 2012

Summary

Phenylalanine 317 in the rubber-degrading enzyme RoxA is crucial for its catalytic activity. Mutating this residue significantly reduces or eliminates the enzyme's ability to cleave polyisoprene, highlighting its role in substrate interaction.

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Area of Science:

Biochemistry
Enzymology
Microbial Biotechnology

Background:

RoxA is an extracellular c-type diheme cytochrome from Xanthomonas sp. strain 35Y.
This enzyme degrades poly(cis-1,4-isoprene), a component of rubber.
The enzyme's structure suggests Phenylalanine 317 (Phe317) is near the active site heme.

Purpose of the Study:

To investigate the role of Phenylalanine 317 (Phe317) in the catalytic activity of RoxA.
To determine if Phe317 is essential for polyisoprene cleavage.

Main Methods:

Site-directed mutagenesis of RoxA to replace Phe317 with other amino acids (tyrosine, tryptophan, leucine, histidine, alanine).
Expression of RoxA variants in a Xanthomonas sp. ΔroxA strain.
Assessing polyisoprene cleavage activity using latex agar assays and purified muteins.
UV-visible spectroscopy to analyze heme presence and ligand binding.

Main Results:

Mutations Phe317Tyr and Phe317Trp rendered RoxA inactive.
Mutations Phe317Leu, Phe317Ala, and Phe317His showed reduced activity (wild type > Leu > Ala > His).
Purified Phe317Ala and Phe317Leu variants had significantly reduced cleavage activity (≈3% and 10%).
Spectroscopic analysis indicated altered interactions with ligand molecules for RoxA muteins.

Conclusions:

Phenylalanine 317 is essential for RoxA's polyisoprene cleavage activity.
This residue plays a critical role in the enzyme's interaction with its substrate.
This study provides the first structure-function analysis of a polyisoprene-cleaving enzyme and identifies a key active site residue.