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Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments.

ABSTRACT: Coproheme decarboxylases (ChdCs) are enzymes responsible for the catalysis of the terminal step in the coproporphyrin-dependent heme biosynthesis pathway. Phylogenetic analyses confirm that the gene encoding for ChdCs is widespread throughout the bacterial world. It is found in monoderm bacteria (Firmicutes, Actinobacteria), diderm bacteria (e. g. Nitrospirae) and also in Archaea. In order to test phylogenetic prediction ChdC representatives from all clades were expressed and examined for their coproheme decarboxylase activity. Based on available biochemical data and phylogenetic analyses a sequence motif (-Y-P-M/F-X-K/R-) is defined for ChdCs. We show for the first time that in diderm bacteria an active coproheme decarboxylase is present and that the archaeal ChdC homolog from Sulfolobus solfataricus is inactive and its physiological role remains elusive. This shows the limitation of phylogenetic prediction of an enzymatic activity, since the identified sequence motif is equally conserved across all previously defined clades.

SUBMITTER: Pfanzagl V

PROVIDER: S-EPMC5993186 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments.

Pfanzagl Vera V Holcik Laurenz L Maresch Daniel D Gorgone Giulia G Michlits Hanna H Furtmüller Paul G PG Hofbauer Stefan S

Archives of biochemistry and biophysics 20180110

Coproheme decarboxylases (ChdCs) are enzymes responsible for the catalysis of the terminal step in the coproporphyrin-dependent heme biosynthesis pathway. Phylogenetic analyses confirm that the gene encoding for ChdCs is widespread throughout the bacterial world. It is found in monoderm bacteria (Firmicutes, Actinobacteria), diderm bacteria (e. g. Nitrospirae) and also in Archaea. In order to test phylogenetic prediction ChdC representatives from all clades were expressed and examined for their ...[more]

PMID: 29331688

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Project description:Background:The CYP152 family member OleTJE from Jeotgalicoccus sp. ATCC 8456 has been well-known to catalyze the unusual one-step decarboxylation of free fatty acids towards the formation of terminal alkenes. Efforts to tune up its decarboxylation activity for better production of biological alkenes have been extensively explored via approaches such as site-directed mutagenesis and electron source engineering, but with limited success. To gain more insights into the decarboxylation mechanism and reaction bifurcation (decarboxylation versus hydroxylation), we turned to an alternative approach to explore the natural CYP152 resources for a better variety of enzyme candidates. Results:We biochemically characterized three new P450 fatty acid decarboxylases including OleTJH, OleTSQ and OleTSA, with respect to their substrate specificity, steady-state kinetics, and salt effects. These enzymes all act as an OleTJE-like fatty acid decarboxylase being able to decarboxylate a range of straight-chain saturated fatty acids (C8-C20) to various degrees. Site-directed mutagenesis analysis to the lower activity P450 enzyme OleTSA revealed a number of key amino acid residues within the substrate-binding pocket (T47F, I177L, V319A and L405I) that are important for delicate substrate positioning of different chain-length fatty acids and thus the decarboxylation versus hydroxylation chemoselectivity, in particular for the mid-chain fatty acids (C8-C12). In addition, the three new decarboxylases exhibited optimal catalytic activity and stability at a salt concentration of 0.5 M, and were thus classified as moderate halophilic enzymes. Conclusion:The P450 fatty acid decarboxylases OleTJE, OleTJH, OleTSQ and OleTSA belong to a novel group of moderate halophilic P450 enzymes. OleTJH from Jeotgalicoccus halophilus shows the decarboxylation activity, kinetic parameters, as well as salt tolerance and stability that are comparable to OleTJE. Site-directed mutagenesis of several key amino acid residues near substrate-binding pocket provides important guidance for further engineering of these P450 fatty acid decarboxylases that hold promising application potential for production of ?-olefin biohydrocarbons.

Project description:Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.

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Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments.

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Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments.

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