**4. Discussion and conclusion**

Many studies have been done on NAD+-dependent *cis*-dihydrodiol dehydrogenases (DD) in bacteria (Jouanneau & Meyer, 2006; Van Herwijnen et al., 2003). In the case of NADP+-

Polyacrylamide Gel Electrophoresis an Important Tool for the

Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms 261

We have shown that on naphthalene, anthracene, phenanthrene or pyrene used as sole carbon source, there exist three different inducible NADP+-dependent dihydrodiol dehydrogenase activities. One of them iNDD, was the only isoenzyme inducible by all aromatic hydrocarbons which presumably is involved in the aromatic hydrocarbon biodegradation pathway in YR-1 strain (Fig. 1). In particular iNDD was capable to use only *cis-*naphthalene-diol as substrate (Fig. 7) suggesting that this enzyme is specific part of the metabolic pathway of the naphthalene; all other activity bands are ADH substrate unspecific It is interesting that iPDD2 has broad substrate specificity when NADP+ was the electron acceptor, suggesting that it could be one of the different dehydrogenases belonging to the microsomal system of alcohol (ethanol) oxidation [MEOS (Krauzova et al., 1985)]. No one of

For iNDD, naphthalene was the best inducer and pyrene the worst. In the case of both iPDD1 and 2, phenanthrene was the only inducer of these enzymes, however pyrene shows a very low inducer effect on iPDD1 (Table 3). The finding of two inducible (iNDD and iPDD1) and one constitutive (DD1) enzymes that uses specifically a dihydrodiol as substrate is in agreement with the number of three possible DD's of predicted function, reported in

With regard to the constitutive dihydrodiol dehydrogenase activities present in YR-1 strain, four of them use only *cis-*naphthalene-diol as substrate: DD1, 2, 3 and 7; DD2, use both *cis*naphthalene-diol and with high efficiency propane-1,2,3-triol, indicating that it can be the glycerol dehydrogenase-1 (iGlcDH1 inducible by 1-decanol) described previously by

Our above-mentioned findings with *M. circinelloides* YR-1 dihydrodiol dehydrogenase activities are indicative of developmental regulation of the different DD's enzymes; this interpretation is supported by following observations: in zymograms for DD's activities when YR-1 was grown in different carbon sources is showed a differential pattern of the activity bands depending of the carbon source used in the culture media. The present results suggest the existence of eleven enzymes with dihydrodiol dehydrogenase activity. Particularly important the DD1 that could be the constitutive DD, and iNDD iPDD1, that could be part of the aromatic hydrocarbon biodegradation pathway in YR-1 strain for naphthalene or the others aromatic hydrocarbon, respectively. Future genomic analysis after isolation of the respective genes should prove the existence of one gene for each constitutive or inducible activity in agreement with the *M. circinelloides* data base prediction. The details of the possible

interaction between alcohols or hydrocarbons metabolism remain to be determined.

100, No. 1-9 (March 2002), pp. 243-255. ISSN: 0273-2289.

Support for this research by Universidad de Guanajuato (México), is gratefully

Alvarado, C. Y., Gutiérrez-Corona, F. & Zazueta-Sandoval, R. (2002). Presence and

physiologic regulation of alcohol oxydase activity in an indigenous fungus isolated from petroleum-contaminated soils. *Applied Biochemistry and Biotechnology*. Vol. 98-

all inducible DD's activities showed to be able to use NAD+ as electron acceptor.

database of *Mucor circinelloides* (Torres-Martínez et al., 2009).

ourselves in YR-1 (Camacho et al., 2010).

**5. Acknowledgments** 

acknowledged.

**6. References** 

dependent *trans*-dihydrodiol dehydrogenases, almost all investigations have been done in mammalian tissues (Carbone et al., 2008; Chang et al, 2009; Chen et al., 2008) but only a few reports about these important enzymes have been done in fungi (Bezalel et al., 1997; Hammel, 1995; Sutherland et al., 1993) particularly in *Phanerochaete chrisosporium* (Bogan & Lamar, 1996; Muheim et al., 1991). At date, there is no any report about the detection of dihydrodiol dehydrogenase activities by means of electrophoretic zymograms in any organism. This methodology represents an interesting approach because in this way it is possible to detect, study and compare the different isoenzymes present in the cell-free extracts of the organism used as enzymatic source. In our own work, YR-1 strain possesses extraordinary metabolic machinery that premises it to survive in a very dangerous place how is a petroleum-contaminated soil.

The results about the localization of DD activities in a differential centrifugation procedure from YR-1 grown in different carbon sources (Table 1), revealed that the activity measured with *cis*-naphthalene-diol as substrate and NADP+ as electron acceptor was only present in the supernatant fractions of each centrifugation speed, suggesting that all DD activity observed must be a soluble enzyme. At date, we cannot discard the possibility that the DD activity could be located in the lumen of some kind of microsomal bodies, because of the drastic ballistic treatment used to homogenize the cells. Actually, we are conducting different experiments employing density gradients and electron transmission microscopy to resolve this question.

Complementary analysis of DD activities by electrophoretic zymograms led us to detect eleven different activities and all of them were NADP+-dependent (Fig. 2) this represents the first report about the detection of DD activities by electrophoretic zymograms, a nondenaturing gel electrophoresis stained with a colored product of the enzymatic reaction.

Of the eleven bands detected, we described five different constitutive DD activities, DD 1-5, since them were observed when D-glucose was the carbon source and only DD-2 was solely induced by this sugar, since the others are induced at least for another carbon source. When *n*-decanol was used as a carbon source, we observed four out of five of the constitutive bands, lacking only the DD-2 band. Its noticeable that only when glycerol, ethanol, npentane and n-hexadecane were the carbon source to grow the fungus, not a single constitutive band was observed, may be due to the fact that these compounds only can be metabolized specifically by the induced enzymes. In glucose grown mycelium, all inducible dihydrodiol dehydrogenase activities were absent suggesting that they could be subject to carbon-catabolite repression (Fig. 2).

Surprisingly all the activities described here as DD are able to use *cis*-naphthalene-diol, since this substrate has been describes as bacterial specific (Cerniglia & Gibson 1977). The substrate reported for eukaryotic cells is the *trans*-naphthalene-diol (Cerniglia 1977).

Phenanthrene was the best inducer since when used as a carbon source four out of six inducible bands were observed, *n*-decanol and naphthalene were the second best inducers since each one led the induction of three different enzymes, sharing the bands denominated iDD1 and iDD2. Also in the case of the inducible enzymes glycerol, n-pentane and nhexadecane were unable to induce any activity. The specific induction of an activity must be due to substrate specificity.

We have shown that on naphthalene, anthracene, phenanthrene or pyrene used as sole carbon source, there exist three different inducible NADP+-dependent dihydrodiol dehydrogenase activities. One of them iNDD, was the only isoenzyme inducible by all aromatic hydrocarbons which presumably is involved in the aromatic hydrocarbon biodegradation pathway in YR-1 strain (Fig. 1). In particular iNDD was capable to use only *cis-*naphthalene-diol as substrate (Fig. 7) suggesting that this enzyme is specific part of the metabolic pathway of the naphthalene; all other activity bands are ADH substrate unspecific

It is interesting that iPDD2 has broad substrate specificity when NADP+ was the electron acceptor, suggesting that it could be one of the different dehydrogenases belonging to the microsomal system of alcohol (ethanol) oxidation [MEOS (Krauzova et al., 1985)]. No one of all inducible DD's activities showed to be able to use NAD+ as electron acceptor.

For iNDD, naphthalene was the best inducer and pyrene the worst. In the case of both iPDD1 and 2, phenanthrene was the only inducer of these enzymes, however pyrene shows a very low inducer effect on iPDD1 (Table 3). The finding of two inducible (iNDD and iPDD1) and one constitutive (DD1) enzymes that uses specifically a dihydrodiol as substrate is in agreement with the number of three possible DD's of predicted function, reported in database of *Mucor circinelloides* (Torres-Martínez et al., 2009).

With regard to the constitutive dihydrodiol dehydrogenase activities present in YR-1 strain, four of them use only *cis-*naphthalene-diol as substrate: DD1, 2, 3 and 7; DD2, use both *cis*naphthalene-diol and with high efficiency propane-1,2,3-triol, indicating that it can be the glycerol dehydrogenase-1 (iGlcDH1 inducible by 1-decanol) described previously by ourselves in YR-1 (Camacho et al., 2010).

Our above-mentioned findings with *M. circinelloides* YR-1 dihydrodiol dehydrogenase activities are indicative of developmental regulation of the different DD's enzymes; this interpretation is supported by following observations: in zymograms for DD's activities when YR-1 was grown in different carbon sources is showed a differential pattern of the activity bands depending of the carbon source used in the culture media. The present results suggest the existence of eleven enzymes with dihydrodiol dehydrogenase activity. Particularly important the DD1 that could be the constitutive DD, and iNDD iPDD1, that could be part of the aromatic hydrocarbon biodegradation pathway in YR-1 strain for naphthalene or the others aromatic hydrocarbon, respectively. Future genomic analysis after isolation of the respective genes should prove the existence of one gene for each constitutive or inducible activity in agreement with the *M. circinelloides* data base prediction. The details of the possible interaction between alcohols or hydrocarbons metabolism remain to be determined.
