BACKGROUND
members of the short-chain dehydrogenase/reductase  superfamily are non-metallo enzymes with molecular masses between  <dig> and  <dig> kda that function in the form of dimers or tetramers  <cit> .
the crystal structures of several sdr proteins have revealed that the protein fold is highly conserved within this superfamily  <cit> .
the sdr tetramers display  <dig> symmetry with two different interfaces between their  <dig> subunits, described by three orthogonal molecular axes, called p, q and r  <cit> .
the most extensive contacts are those spanning the q-axis and involve two helices, αe and αf.
in most homodimeric sdrs for which the structure is known, dimerization also occurs across this interface.
so far, only one sdr protein, the 3α-hydroxysteroid dehydrogenase/carbonyl reductase  from comamonas testosteroni , has been shown to differ in its mode of dimerization.
in this case, dimerization takes place across the p-axis interface, formed mainly by the αg helices and by interactions between the βg strands of two neighbouring subunits  <cit> .
to date, only two monomeric structures, those of porcine testicular carbonyl reductase   and human carbonyl reductase  , are known within the sdr superfamily  <cit> .
although there are about three thousand members of the sdr superfamily in the species studied  <cit> , 17β-hsd from the fungus cochliobolus lunatus   <cit>  is currently the only fungal hsd member that has been characterized.
the enzyme has been purified, and also cloned and expressed in e. coli  <cit> .
under native conditions, both recombinant  <cit>  and natural  <cit>  17β-hsdcl form dimers.
17β-hsdcl is homologous to fungal reductases: to versicolorin reductases from aspergillus parasiticus and emericella nidulans, which are involved in aflatoxin biosynthesis; and to the  <dig> ,8-trihydroxynaphthalene reductases and  <dig> , <dig> -tetrahydroxynaphthalene reductases  from magnaporthe grisea, ophiostoma floccosum and other fungi, which are involved in melanin biosynthesis  <cit> .
3hnr and 4hnr catalyze an essential reaction in the biosynthesis of melanin, a virulence factor of phyto-pathogenic fungi, as well as of fungi pathogenic to humans  <cit> .
these enzymes are the biochemical targets of several commercially important fungicides that are used to prevent blast disease in rice plants  <cit> .
the study of the fungal 17β-hsd will, therefore, contribute to a better understanding of the functionality of these homologous fungal enzymes that are targets for the design of novel antifungal agents.
as fungal 17β-hsd exhibits about 30% amino acid identity to human 17β-hsd types  <dig> and  <dig>  <cit> , this could also lead to an understanding of the mechanisms of catalysis in human hsds, which are implicated in the development of steroid-dependent forms of cancer, polycystic kidney disease, regulation of blood pressure, alzheimer's disease and obesity  <cit> .
a detailed understanding of the individual amino acids that are important for dimerization could enable the design of compounds that would specifically prevent dimerization, and consequently the enzyme activity of the sdr proteins, and should therefore serve as new types of drugs  <cit> .
dimerization of the sdr proteins has already been studied in drosophila alcohol dehydrogenase , where ethyl methanesulphonate-induced null mutants were prepared and examined.
among the mutants studied, the inactive mutant ala159thr did not form a stable homodimer, suggesting that ala <dig> is important for hydrophobic interactions that stabilize the adh dimer  <cit> .
dimerization has also been studied in human 17β-hsd type  <dig>  an enzyme that has a high affinity for dimer formation  and consequently exists only as a dimer in vitro  <cit> .
the 17β-hsd type  <dig> mutations leu111glu/val113phe and ala170glu+phe <dig> abolished its activity and changed the folding of the enzyme.
both mutated proteins formed aggregates with apparent molecular weights of more than  <dig> kda; monomeric or dimeric forms of the enzyme were not seen  <cit> .
based on a comparison of the structures of fungal 17β-hsd  <cit>   and the homotetrameric 3hnr from magnaporthe grisea   <cit> , we investigated the dimerization of 17β-hsdcl by site-directed mutagenesis.
we have identified the secondary structure elements and the amino acids essential for dimerization and have demonstrated the relationship between dimerization and enzyme activity.
RESULTS
structural comparisons indicate that his <dig> and arg <dig> are involved in dimerization
the sequence alignments and structural comparison of the three-dimensional models of fungal 17β-hsd  <cit> , supported by our preliminary crystallographic data  <cit> , with the homologous 3hnr , aligned without gaps between residue number  <dig> to  <dig> in 17β-hsdcl and residues  <dig> to  <dig> in 3hnr , identified strong hydrophobic and aromatic interactions across the q-axis  and potential salt bridges/h-bonds between the αe and αf helices of the two subunits, all of which may stabilize dimerization .
one salt bridge can be formed between arg <dig> and glu <dig> and/or asp <dig> from the αe helices, while his <dig>  which is positioned in front of the αe helix, can form an h-bond with asp <dig> from the αf helix .
the interactions between the p-axis-related subunits are not so extensive and the surface amino acids are less hydrophobic than those of tetrameric 3hnr .
to test our hypothesis that dimerization takes place across the q-axis, the his111ala, his111leu and arg129asp mutants were prepared with the aim of selectively omitting one h-bond  and of introducing electrostatic repulsion between the monomers .
the his111leu and arg129asp mutations each result in monomeric proteins
the purities of the expressed proteins were checked by sds-page, where all of the proteins appeared as bands with the expected molecular mass of  <dig> kda and were approx.
90% homogenous.
the solution molecular mass was determined by gel filtration and by non-denaturing page.
his111ala-17β-hsdcl remained a dimer, as with the wild-type protein, while the other two mutations, his111leu and arg129asp, resulted in monomers .
the dimers eluted at the retention time of albumin with a molecular weight of  <dig> kda, and the monomers eluted at a time similar to that of carbonic anhydrase  .
differences in mobility of monomeric mutants compared to the dimeric proteins was also observed on the native page .
monomer secondary structure is conserved, although the proteins differ in their thermal denaturation characteristics
the secondary structures of the wild-type and mutant enzymes were analysed through circular dichroism spectroscopy.
this method revealed similar conformations for all the proteins .
the thermal denaturation was measured by following the change of the ellipticity at  <dig> nm as a function of the temperature.
two behaviours were recognized, that of the wild-type protein and the his111ala mutant, and that of the two monomeric mutants, his111leu and arg129asp.
the melting curves of the wild-type protein and the his111ala mutant were sigmoidal, revealing a cooperative denaturation.
the wild-type protein had a tm of 44°c, and the his111ala mutant, of 37°c.
the forms of the melting curves of the other two mutants suggested lower cooperativities of denaturation.
the melting points were 48°c for the his111leu mutant and 50°c for arg129asp  .
kinetic constants for the reduction and oxidation of the substrates 4-estrene- <dig> -dione and 4-estrene-17β-ol-3-one   that were catalyzed by the wild-type and mutant 17β-hsdcl in the presence of the coenzymes nadph and nadp+ , at ph  <dig> and 25°c.
the temperature midpoints  are also given.
the coenzyme binds to the monomeric mutants
we measured the formation of the binary complexes of e•nadp+ for the wild-type and mutant enzymes.
the fluorescence of all of the proteins was quenched by the addition of the coenzyme, but the kd values differed.
the kd value for the dimeric mutant his111ala increased 30-fold, and those for the his111leu and arg129asp monomers increased 100-fold and 60-fold, respectively, when compared with the wild-type enzyme .
these results support the circular dichroism data, suggesting only minor changes in the coenzyme binding site, even in the monomeric enzymes.
the his111leu and arg129asp mutants are not active on the substrates
the oxidation of 4-estrene-17β-ol-3-one and the reduction of 4-estrene- <dig> -dione by the wild-type and mutant proteins in the presence of nadp+ and nadph showed enzyme activities for the wild-type protein and for the his111ala mutant, but no detectable activities for the monomeric his111leu and arg129asp proteins within a  <dig> s sweep.
although the his111ala mutant was active, its kinetic constants differed from those of the wild-type protein .
the km values for both of the coenzymes, nadp+ and nadph, were increased in the his111ala mutant , while the kcat values remained similar .
the specificity constants, kcat/km, for nadp+ and nadph, were 58-fold and 12-fold lower, respectively, when compared to those for the wild-type enzyme .
since we could not detect any enzyme activities with the monomeric mutants within the  <dig> s sweep, we also tested the enzyme activities by tlc analysis of the products after 24-h incubations of the proteins with 4-estrene-17β-ol-3-one and nadp+.
again, the wild-type and his111ala mutant proteins converted this substrate to equilibrium concentrations, while the his111leu and arg129asp mutant proteins did not .
discussion
the dimerization of fungal 17β-hsd occurs across the q-axis
among the tens of sdr enzymes for which the three-dimensional structures have been resolved, only two are known to be active in solution as monomers   <cit> .
we have shown previously, that the wild-type 17β-hsdcl acts as dimer  <cit> .
in order to influence the formation of this dimer, we substituted two surface amino acids which should not, per se, prevent either the folding or the activity.
to avoid possible cooperative phenomena, we prepared mutants with single substitutions, yet we knew that in the dimer the effects would be doubled.
inspection of the three-dimensional structural model of 17β-hsdcl  <cit>  supported by our preliminary crystallographic data  <cit>  revealed that his <dig> from each subunit could form an h-bond with asp <dig> from another subunit, while arg <dig> could form salt bridge with two negatively charged candidates, asp <dig> and glu <dig>  the importance of these interactions was confirmed directly with three mutations: his111ala/leu and arg129asp.
the his111ala mutation resulted in dimers, while that of his111leu prevented the formation of an h-bond as well as the formation of a dimer, most probably because of the steric hindrance introduced by leu .
substitution of arg <dig> by asp results in electrostatic repulsion between the monomers, and simple calculations revealed that the two abolished salt-bridges result in an approximately 1014-fold higher dimer dissociation constant , thus preventing dimerization.
on the other hand, if all of the binding energy were realized in the affinity change caused by two h-bonds, as in the his <dig> mutants, only a half of the above change would be expected.
it appears that this is still not enough to prevent dimerization , unless the steric component is added .
since both of the substituted residues are the members of the αe helix, these results strongly suggest that dimerization of fungal 17β-hsd takes place across the q-axis.
the denaturation of monomers occurs at higher temperatures
the results showed that the thermal denaturation of the wild-type protein and the dimeric his111ala mutant is a cooperative process that involves the disruption of the dimers and the denaturation of the monomers.
compared with the wild-type protein, denaturation of this mutant occurred at a lower temperature .
the results indicated that the mutation introduced causes a decrease in the thermal stability of the protein, and that the his in position  <dig> is involved in interactions that stabilize the dimer.
the higher melting temperature of the two monomeric mutants, his111leu and arg129asp, may be associated with a higher stability of the monomers and with a decreased flexibility of their structure in comparison with the dimers.
a higher flexibility may be needed to rearrange the conformation after binding of the cofactor, thus enabling proper binding of the substrate, and this may also explain the loss of activity of the his111leu and arg129asp mutants.
additionally, the icebergs of solvent  around the exposed hydrophobic groups of the αe and αf helices may contribute to the higher tm.
as the temperature is raised, the icebergs melt and their latent heat of fusion contributes significantly to the specific heat capacity  of the denatured state  <cit> .
dimerization is crucial for enzyme activity
the substitution of his <dig> by ala at the q-interface resulted in higher kd values and lower specificity constants  of this dimer when compared with the wild-type enzyme, while the substitution of his <dig> by leu and of arg <dig> by asp resulted in inactive monomers.
these results indicate that h-bonds/salt bridges contribute to both the specific dimerization and the enzyme activity.
the stability of the q-interface, which is composed of the αe and αf helices, is crucial for enzyme catalysis in the sdr enzymes since two catalytic residues, tyr and lys, reside in the interior side of the αf helix, one catalytic residue, asn, resides in the interior side of the αe helix, while ser is positioned in the small αef helix, between the βe strand and the αf helix  <cit> .
the proper orientation of these catalytically important helices assures the proper orientation  of the catalytic amino acids to the nicotinamide ring and the substrate, enabling the hydride transfer.
the investigation of another sdr member, glucose dehydrogenase  from bacillus megaterium iwg <dig>   <cit> , supports our hypothesis.
glcdh functions as a tetramer and its crystal structure has revealed weaker hydrophobic interactions and a lack of salt bridges in the q-interface.
for these reasons, the glcdh tetramer dissociates into inactive monomers at ph  <dig> , but reversibly associates into the fully active tetramer at ph  <dig> .
this is not the case for our fungal enzyme, which is active in the ph range of  <dig>  to  <dig>   <cit> .
our results and those of others have thus demonstrated that only upon dimerization can the key catalytic residues be brought into the positions that enable the substrate to be converted.
since both of the mutant monomers show no enzymatic activity despite their conserved secondary structures and the positive coenzyme binding , it appears that the enzyme-coenzyme ) binary complex undergoes conformational changes before the substrate binding can occur.
such a conformational adaptation has previously been proposed for 3α-hsd from the sdr superfamily and for several members of the aldo-keto reductase superfamily  <cit> .
in these proteins, the binding of nadp involves two events: an initial formation of a loose complex ), followed by a conformational change in the enzyme structure that leads to a tightly bound complex )  <cit> .
our data are in agreement with his hypothesis.
dimerization via the q-axis is not possible in 3α-hsd/cr, ptcr and cbr1
on comparing the crystal structures of dimeric and tetrameric sdrs with those of 3αhsd/cr, where dimerization takes place via a p-axis interface  <cit> , and of ptcr and cbr <dig>  the only monomeric enzyme structures of the sdr superfamily  <cit> , it becomes evident that in all three structures, interactions across the q-axis are blocked by the presence of a predominantly α-helical subdomain.
in these three proteins, this  <dig>   <dig> or  <dig> amino-acid insertion into the classical rossman-fold motif between strand βe and helix αf, respectively, prevents the formation of a four-helix bundle, while at the same time it masks, and in this manner stabilizes, the αe and αf helices.
to determine whether the lack of this subdomain leads to the formation of a homotetramer, a redesign of this interface was recently performed in the dimeric 3α-hsd/cr, and further enzymatic characterization is currently in progress  <cit> .
this insertion is not present in other sdrs that can stabilize the αe and αf helices by dimerization via the q-axis interface.
therefore, again, dimerization appears to be crucial for the stability of the αe and αf helices in the fungal 17β-hsd as well as in other sdr proteins, except 3α-hsd/cr, ptcr and cbr <dig>
selective prevention of dimerization may enable the development of a new type of drug
a detailed understanding of the importance of specific residues for dimerization should enable the design of compounds that can prevent the dimerization and thus serve as a new type of drug.
this proposal is supported by the finding that the residues involved in subunit interactions differ considerably between the enzymes.
recently, caffrey et al.
examined the difference in conservation between the protein-protein interfaces and the rest of the protein surfaces for a set of  <dig> proteins from different protein superfamilies  <cit> .
a surface-patch analysis showed that this interface is rarely significantly more conserved than other surface patches  <cit> .
although almost all of the sdr proteins span across the q-axis interface, the residues involved in the dimerization are not conserved.
subunit interactions at the αe-αe contact regions were compared in  <dig> members of the sdr superfamily   <cit> , whereby different modes of subunit interactions were shown in these proteins.
therefore, the concept that the selective prevention of dimerization in the sdr proteins will lead to the development of a new type of inhibitors is reasonable.
CONCLUSIONS
fungal 17β-hsd is a dimeric member of the sdr superfamily.
we have shown by site-directed mutagenesis and structure/function analysis that the dimerization takes place across the q-axis and involves the αe and αf helices of both subunits.
neighbouring subunits are connected through hydrophobic interactions, h-bonds and salt bridges involving amino-acid residues his <dig> and arg <dig>  the substitution of these amino acid residues leads to inactive monomers with conserved secondary structures that are still able to bind the coenzyme.
our results indicate that the stability of the αe and αf helices that include catalytic amino acid residues is crucial for enzyme activity.
although oligomerization of the sdr proteins has already been studied in drosophila alcohol dehydrogenase and human 17β-hsd type  <dig>  our study is the first that has prepared monomeric mutants in sufficient quantities and qualities for structure/function analysis.

 members of the short-chain dehydrogenase/reductase superfamily are non-metallo enzymes with molecular masses between and kda that function in the form of dimers or tetramers. the crystal structures of several sdr proteins have revealed that the protein fold is highly conserved within this superfamily. the sdr tetramers display symmetry with two different interfaces between their subunits, described by three orthogonal molecular axes, called p, q and r. the most extensive contacts are those spanning the q-axis and involve two helices, αe and αf. in most homodimeric sdrs for which the structure is known, dimerization also occurs across this interface. so far, only one sdr protein, the 3α-hydroxysteroid dehydrogenase/carbonyl reductase from comamonas testosteroni, has been shown to differ in its mode of dimerization. in this case, dimerization takes place across the p-axis interface, formed mainly by the αg helices and by interactions between the βg strands of two neighbouring subunits. to date, only two monomeric structures, those of porcine testicular carbonyl reductase and human carbonyl reductase, are known within the sdr superfamily. although there are about three thousand members of the sdr superfamily in the species studied, 17β-hsd from the fungus cochliobolus lunatus is currently the only fungal hsd member that has been characterized. the enzyme has been purified, and also cloned and expressed in e. coli. under native conditions, both recombinant and natural 17β-hsdcl form dimers. 17β-hsdcl is homologous to fungal reductases: to versicolorin reductases from aspergillus parasiticus and emericella nidulans, which are involved in aflatoxin biosynthesis; and to the,8-trihydroxynaphthalene reductases and, -tetrahydroxynaphthalene reductases from magnaporthe grisea, ophiostoma floccosum and other fungi, which are involved in melanin biosynthesis. 3hnr and 4hnr catalyze an essential reaction in the biosynthesis of melanin, a virulence factor of phyto-pathogenic fungi, as well as of fungi pathogenic to humans. these enzymes are the biochemical targets of several commercially important fungicides that are used to prevent blast disease in rice plants. the study of the fungal 17β-hsd will, therefore, contribute to a better understanding of the functionality of these homologous fungal enzymes that are targets for the design of novel antifungal agents. as fungal 17β-hsd exhibits about 30% amino acid identity to human 17β-hsd types and, this could also lead to an understanding of the mechanisms of catalysis in human hsds, which are implicated in the development of steroid-dependent forms of cancer, polycystic kidney disease, regulation of blood pressure, alzheimer's disease and obesity. a detailed understanding of the individual amino acids that are important for dimerization could enable the design of compounds that would specifically prevent dimerization, and consequently the enzyme activity of the sdr proteins, and should therefore serve as new types of drugs. dimerization of the sdr proteins has already been studied in drosophila alcohol dehydrogenase, where ethyl methanesulphonate-induced null mutants were prepared and examined. among the mutants studied, the inactive mutant ala159thr did not form a stable homodimer, suggesting that ala is important for hydrophobic interactions that stabilize the adh dimer. dimerization has also been studied in human 17β-hsd type an enzyme that has a high affinity for dimer formation and consequently exists only as a dimer in vitro. the 17β-hsd type mutations leu111glu/val113phe and ala170glu+phe abolished its activity and changed the folding of the enzyme. both mutated proteins formed aggregates with apparent molecular weights of more than kda; monomeric or dimeric forms of the enzyme were not seen. based on a comparison of the structures of fungal 17β-hsd and the homotetrameric 3hnr from magnaporthe grisea, we investigated the dimerization of 17β-hsdcl by site-directed mutagenesis. we have identified the secondary structure elements and the amino acids essential for dimerization and have demonstrated the relationship between dimerization and enzyme activity.  structural comparisons indicate that his and arg are involved in dimerization the sequence alignments and structural comparison of the three-dimensional models of fungal 17β-hsd, supported by our preliminary crystallographic data, with the homologous 3hnr, aligned without gaps between residue number to in 17β-hsdcl and residues to in 3hnr, identified strong hydrophobic and aromatic interactions across the q-axis and potential salt bridges/h-bonds between the αe and αf helices of the two subunits, all of which may stabilize dimerization. one salt bridge can be formed between arg and glu and/or asp from the αe helices, while his which is positioned in front of the αe helix, can form an h-bond with asp from the αf helix. the interactions between the p-axis-related subunits are not so extensive and the surface amino acids are less hydrophobic than those of tetrameric 3hnr. to test our hypothesis that dimerization takes place across the q-axis, the his111ala, his111leu and arg129asp mutants were prepared with the aim of selectively omitting one h-bond and of introducing electrostatic repulsion between the monomers. the his111leu and arg129asp mutations each result in monomeric proteins the purities of the expressed proteins were checked by sds-page, where all of the proteins appeared as bands with the expected molecular mass of kda and were approx. 90% homogenous. the solution molecular mass was determined by gel filtration and by non-denaturing page. his111ala-17β-hsdcl remained a dimer, as with the wild-type protein, while the other two mutations, his111leu and arg129asp, resulted in monomers. the dimers eluted at the retention time of albumin with a molecular weight of kda, and the monomers eluted at a time similar to that of carbonic anhydrase. differences in mobility of monomeric mutants compared to the dimeric proteins was also observed on the native page. monomer secondary structure is conserved, although the proteins differ in their thermal denaturation characteristics the secondary structures of the wild-type and mutant enzymes were analysed through circular dichroism spectroscopy. this method revealed similar conformations for all the proteins. the thermal denaturation was measured by following the change of the ellipticity at nm as a function of the temperature. two behaviours were recognized, that of the wild-type protein and the his111ala mutant, and that of the two monomeric mutants, his111leu and arg129asp. the melting curves of the wild-type protein and the his111ala mutant were sigmoidal, revealing a cooperative denaturation. the wild-type protein had a tm of 44°c, and the his111ala mutant, of 37°c. the forms of the melting curves of the other two mutants suggested lower cooperativities of denaturation. the melting points were 48°c for the his111leu mutant and 50°c for arg129asp. kinetic constants for the reduction and oxidation of the substrates 4-estrene- -dione and 4-estrene-17β-ol-3-one that were catalyzed by the wild-type and mutant 17β-hsdcl in the presence of the coenzymes nadph and nadp+, at ph and 25°c. the temperature midpoints are also given. the coenzyme binds to the monomeric mutants we measured the formation of the binary complexes of e•nadp+ for the wild-type and mutant enzymes. the fluorescence of all of the proteins was quenched by the addition of the coenzyme, but the kd values differed. the kd value for the dimeric mutant his111ala increased 30-fold, and those for the his111leu and arg129asp monomers increased 100-fold and 60-fold, respectively, when compared with the wild-type enzyme. these results support the circular dichroism data, suggesting only minor changes in the coenzyme binding site, even in the monomeric enzymes. the his111leu and arg129asp mutants are not active on the substrates the oxidation of 4-estrene-17β-ol-3-one and the reduction of 4-estrene- -dione by the wild-type and mutant proteins in the presence of nadp+ and nadph showed enzyme activities for the wild-type protein and for the his111ala mutant, but no detectable activities for the monomeric his111leu and arg129asp proteins within a s sweep. although the his111ala mutant was active, its kinetic constants differed from those of the wild-type protein. the km values for both of the coenzymes, nadp+ and nadph, were increased in the his111ala mutant, while the kcat values remained similar. the specificity constants, kcat/km, for nadp+ and nadph, were 58-fold and 12-fold lower, respectively, when compared to those for the wild-type enzyme. since we could not detect any enzyme activities with the monomeric mutants within the s sweep, we also tested the enzyme activities by tlc analysis of the products after 24-h incubations of the proteins with 4-estrene-17β-ol-3-one and nadp+. again, the wild-type and his111ala mutant proteins converted this substrate to equilibrium concentrations, while the his111leu and arg129asp mutant proteins did not. discussion the dimerization of fungal 17β-hsd occurs across the q-axis among the tens of sdr enzymes for which the three-dimensional structures have been resolved, only two are known to be active in solution as monomers. we have shown previously, that the wild-type 17β-hsdcl acts as dimer. in order to influence the formation of this dimer, we substituted two surface amino acids which should not, per se, prevent either the folding or the activity. to avoid possible cooperative phenomena, we prepared mutants with single substitutions, yet we knew that in the dimer the effects would be doubled. inspection of the three-dimensional structural model of 17β-hsdcl supported by our preliminary crystallographic data revealed that his from each subunit could form an h-bond with asp from another subunit, while arg could form salt bridge with two negatively charged candidates, asp and glu the importance of these interactions was confirmed directly with three mutations: his111ala/leu and arg129asp. the his111ala mutation resulted in dimers, while that of his111leu prevented the formation of an h-bond as well as the formation of a dimer, most probably because of the steric hindrance introduced by leu. substitution of arg by asp results in electrostatic repulsion between the monomers, and simple calculations revealed that the two abolished salt-bridges result in an approximately 1014-fold higher dimer dissociation constant, thus preventing dimerization. on the other hand, if all of the binding energy were realized in the affinity change caused by two h-bonds, as in the his mutants, only a half of the above change would be expected. it appears that this is still not enough to prevent dimerization, unless the steric component is added. since both of the substituted residues are the members of the αe helix, these results strongly suggest that dimerization of fungal 17β-hsd takes place across the q-axis. the denaturation of monomers occurs at higher temperatures the results showed that the thermal denaturation of the wild-type protein and the dimeric his111ala mutant is a cooperative process that involves the disruption of the dimers and the denaturation of the monomers. compared with the wild-type protein, denaturation of this mutant occurred at a lower temperature. the results indicated that the mutation introduced causes a decrease in the thermal stability of the protein, and that the his in position is involved in interactions that stabilize the dimer. the higher melting temperature of the two monomeric mutants, his111leu and arg129asp, may be associated with a higher stability of the monomers and with a decreased flexibility of their structure in comparison with the dimers. a higher flexibility may be needed to rearrange the conformation after binding of the cofactor, thus enabling proper binding of the substrate, and this may also explain the loss of activity of the his111leu and arg129asp mutants. additionally, the icebergs of solvent around the exposed hydrophobic groups of the αe and αf helices may contribute to the higher tm. as the temperature is raised, the icebergs melt and their latent heat of fusion contributes significantly to the specific heat capacity of the denatured state. dimerization is crucial for enzyme activity the substitution of his by ala at the q-interface resulted in higher kd values and lower specificity constants of this dimer when compared with the wild-type enzyme, while the substitution of his by leu and of arg by asp resulted in inactive monomers. these results indicate that h-bonds/salt bridges contribute to both the specific dimerization and the enzyme activity. the stability of the q-interface, which is composed of the αe and αf helices, is crucial for enzyme catalysis in the sdr enzymes since two catalytic residues, tyr and lys, reside in the interior side of the αf helix, one catalytic residue, asn, resides in the interior side of the αe helix, while ser is positioned in the small αef helix, between the βe strand and the αf helix. the proper orientation of these catalytically important helices assures the proper orientation of the catalytic amino acids to the nicotinamide ring and the substrate, enabling the hydride transfer. the investigation of another sdr member, glucose dehydrogenase from bacillus megaterium iwg, supports our hypothesis. glcdh functions as a tetramer and its crystal structure has revealed weaker hydrophobic interactions and a lack of salt bridges in the q-interface. for these reasons, the glcdh tetramer dissociates into inactive monomers at ph, but reversibly associates into the fully active tetramer at ph. this is not the case for our fungal enzyme, which is active in the ph range of to. our results and those of others have thus demonstrated that only upon dimerization can the key catalytic residues be brought into the positions that enable the substrate to be converted. since both of the mutant monomers show no enzymatic activity despite their conserved secondary structures and the positive coenzyme binding, it appears that the enzyme-coenzyme ) binary complex undergoes conformational changes before the substrate binding can occur. such a conformational adaptation has previously been proposed for 3α-hsd from the sdr superfamily and for several members of the aldo-keto reductase superfamily. in these proteins, the binding of nadp involves two events: an initial formation of a loose complex ), followed by a conformational change in the enzyme structure that leads to a tightly bound complex ). our data are in agreement with his hypothesis. dimerization via the q-axis is not possible in 3α-hsd/cr, ptcr and cbr1 on comparing the crystal structures of dimeric and tetrameric sdrs with those of 3αhsd/cr, where dimerization takes place via a p-axis interface, and of ptcr and cbr the only monomeric enzyme structures of the sdr superfamily, it becomes evident that in all three structures, interactions across the q-axis are blocked by the presence of a predominantly α-helical subdomain. in these three proteins, this or amino-acid insertion into the classical rossman-fold motif between strand βe and helix αf, respectively, prevents the formation of a four-helix bundle, while at the same time it masks, and in this manner stabilizes, the αe and αf helices. to determine whether the lack of this subdomain leads to the formation of a homotetramer, a redesign of this interface was recently performed in the dimeric 3α-hsd/cr, and further enzymatic characterization is currently in progress. this insertion is not present in other sdrs that can stabilize the αe and αf helices by dimerization via the q-axis interface. therefore, again, dimerization appears to be crucial for the stability of the αe and αf helices in the fungal 17β-hsd as well as in other sdr proteins, except 3α-hsd/cr, ptcr and cbr selective prevention of dimerization may enable the development of a new type of drug a detailed understanding of the importance of specific residues for dimerization should enable the design of compounds that can prevent the dimerization and thus serve as a new type of drug. this proposal is supported by the finding that the residues involved in subunit interactions differ considerably between the enzymes. recently, caffrey et al. examined the difference in conservation between the protein-protein interfaces and the rest of the protein surfaces for a set of proteins from different protein superfamilies. a surface-patch analysis showed that this interface is rarely significantly more conserved than other surface patches. although almost all of the sdr proteins span across the q-axis interface, the residues involved in the dimerization are not conserved. subunit interactions at the αe-αe contact regions were compared in members of the sdr superfamily, whereby different modes of subunit interactions were shown in these proteins. therefore, the concept that the selective prevention of dimerization in the sdr proteins will lead to the development of a new type of inhibitors is reasonable.  fungal 17β-hsd is a dimeric member of the sdr superfamily. we have shown by site-directed mutagenesis and structure/function analysis that the dimerization takes place across the q-axis and involves the αe and αf helices of both subunits. neighbouring subunits are connected through hydrophobic interactions, h-bonds and salt bridges involving amino-acid residues his and arg the substitution of these amino acid residues leads to inactive monomers with conserved secondary structures that are still able to bind the coenzyme. our results indicate that the stability of the αe and αf helices that include catalytic amino acid residues is crucial for enzyme activity. although oligomerization of the sdr proteins has already been studied in drosophila alcohol dehydrogenase and human 17β-hsd type our study is the first that has prepared monomeric mutants in sufficient quantities and qualities for structure/function analysis. 
we have investigated here which secondary structure elements are involved in the dimerization of 17β-hsdcl and examined the importance of dimerization for the enzyme activity. neighbouring subunits are connected through hydrophobic interactions, h-bonds and salt bridges involving amino acid residues his and arg since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. the three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of nadp+, but both monomers showed no enzymatic activity. circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. a detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor. sdr proteins usually function as dimers or tetramers and 17β-hsdcl is also a homodimer under native conditions. sequence similarity with trihydroxynaphthalene reductase from magnaporthe grisea indicated that arg and his from the αe-helices interact with the asp glu and asp residues from the αe and αf-helices of the neighbouring subunit. we have shown by site-directed mutagenesis and structure/function analysis that 17β-hsdcl dimerization involves the αe and αf helices of both subunits. 17β-hydroxysteroid dehydrogenase from the fungus cochliobolus lunatus is a member of the short-chain dehydrogenase/reductase superfamily.
 17β-hydroxysteroid dehydrogenase from the fungus cochliobolus lunatus is a member of the short-chain dehydrogenase/reductase superfamily. sdr proteins usually function as dimers or tetramers and 17β-hsdcl is also a homodimer under native conditions.  we have investigated here which secondary structure elements are involved in the dimerization of 17β-hsdcl and examined the importance of dimerization for the enzyme activity. sequence similarity with trihydroxynaphthalene reductase from magnaporthe grisea indicated that arg and his from the αe-helices interact with the asp glu and asp residues from the αe and αf-helices of the neighbouring subunit. the arg129asp and his111leu mutations both rendered 17β-hsdcl monomeric, while the mutant 17β-hsdcl-his111ala was dimeric. circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. the three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of nadp+, but both monomers showed no enzymatic activity.  we have shown by site-directed mutagenesis and structure/function analysis that 17β-hsdcl dimerization involves the αe and αf helices of both subunits. neighbouring subunits are connected through hydrophobic interactions, h-bonds and salt bridges involving amino acid residues his and arg since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. a detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor. 