This study describes for the first time the cloning, sequencing, and expressing the gene for a eukaryotic PAD in E. coli
. CgPAD exhibited very low sequence similarity to reported functional PADs with 24-39% identity. CgPAD showed 100% amino acid sequence identity to a hypothetical protein (EDK35930; locus tag PGUG_00028) in the genome of the yeast M. guilliermondii
ATCC 6260 (AAFM00000000), and moderate similarity (51-56% identity) to the internal sequences of hypothetical proteins of unknown function in the genomes of fungi including the genera Verticillium, Neosartorya, Aspergillus, Schizophyllum, Ustilago, Sporisorium, Nectria, Gibberella
, and Penicillium
(data not shown). Notably, CgPAD exhibited sequence similarity to PAD1 (YDR538W) with less than 14% identity and essentially no homology with FDC1 (YDR539W) isolated from S. cerevisiae
[Clausen et al. 1994
,Mukai et al. 2010
There was a possibility that either Met57 or Met103 in CgPAD was located in the active-site pocket and oxidized to methionine sulfoxide during growth or purification. Accordingly, the massive sulfoxide group of the oxidized Met residue in the CgPAD might hinder the entry of FA (4-hydroxy-3-methoxycinnamic acid) due to its 3-methoxy group, but not PCA (4-hydroxycinnamic acid), to the active-site pocket. To understand the fluctuation of the ratio of decarboxylation toward FA to PCA of CgPAD, we constructed a model structure of the enzyme and replaced the Met57 residue located at the entrance of the pocket with non-oxidizable amino acids by site-directed mutagenesis. However, a mutant enzyme (M57L) did not increase the decarboxylation ratio of FA to PCA, for instance. This may exclude the possibility that oxidation of Met57, close to the catalytic residues Arg60 and Glu82, alters the decarboxylation ratio of FA to PCA. A single Cys residue at position 66 could be responsible for the alteration of CgPAD activity. However, in the model of CgPAD we built, the Cys66 residue is located deeper in the active-site pocket and faced on the other side and far distant (7.2 Å) from the indole ring of Trp80 which might interact with Glu82 (data not shown). Essentially, conversion of cysteine to cysteic acid during purification steps is unlikely because cysteine is oxidized by strong chemical oxidants.
In this study, the activities toward substrates of the M57T and M57A mutants, together with M57L mutant, were found to be much lower than those of the native and recombinant wild-type forms of CgPAD. This result suggests that Met57 is one of the substrate-binding residues in the catalysis of CgPAD. In support of our view, one of substrate-binding residues, Leu45, in the Enterobacter
PAD [Gu et al. 2011b
] is conserved as Met at the corresponding positions in the aligned bacterial enzymes. The corresponding residue in CgPAD is Met57 in the model structure of CgPAD (see Additional file 4
Unexpectedly, we found that the rate of FA decarboxylation activity, but not PCA decarboxylation activity, of CgPAD was accelerated by DTT, 2-mercaptoethanol, cysteine (both L- and D-forms), and DL-homocysteine, which are antioxidants and/or reducing reagents. However, these chemical reagents cannot reduce oxidized Met residues (methionine sulfoxide and methionine sulfonate) in protein molecules. Furthermore, L-cysteine and L-homocysteine are involved in the trans-sulfurization of amino acid metabolism (e.g., [Brosnan and Brosnan 2006
]), both of which intracellular concentrations are very lowered by strict regulatory control. These results exclude the possibility that these amino acids are the physiological activator for CgPAD.
Finally, we found that an amino thiol-like endogenous factor in the ultrafiltrate of the C. guilliermondii cell-free extract drastically enhanced the FA decarboxylation activity. The kinetic data indicate that the ultrafiltrate increases the maximal activity toward FA without altering of the affinity to the substrate. These findings led us to conclude that a true activator for FA decarboxylation activity is inherently present in the C. guilliermondii cells. This also shows that the true activator was removed during the enzyme purification. Such a catalytic nature has never been reported in the literature. Identification of the structure of the endogenous activator would explain the novel catalytic feature of CgPAD and contribute to the clarification of physiological role of PADs in some yeast cells. It is interesting to examine whether such activation of eukaryotic PADs is observed by ultrafiltrates of prokaryotes and vice versa.
[Rodríguez et al. (2010)
] clarified by site-directed mutagenesis of Arg48 and Glu71 in LpPAD that the entrance region, particularly the β1-β2 and β3-β4 loops, adopted a distinct closed conformation that decreased the opening of the active-site cavity. Possible subsite residues Tyr30 and Tyr32 and catalytic residue Glu82 along with Met57 of CgPAD are located on the β1-β2 loop and β3-β4 loop, respectively (see Additional file 4
A). It is possible that the physiological activator in the ultrafiltrate and/or the tested thiol compounds induce conformational change of the loops so that the entry of FA is much easier than those of PCA and CA. CgPAD exhibits low sequence similarity to LpPAD of known structure, and we are now crystalizing CgPAD to solve its X-ray structure.