Communication Vol. 269, No. 16 , ssue of April 22 , p. 11695-11698, 1994 T H E JOURNAL F Blornlc.4~ CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Opposite Stereospecificity of Tw o Tropinone Reductases Is Conferred by the Substrate-binding Sites* (Recei ved for publicatio n, Ja nu ary 21, 1994) Keiji Nakajima, Takashi Hashimoto$, and Yasuyuki Yamada From the Department o f Agricultural Chemistry, Faculty o f Agriculture, Kyoto University, Kyoto 606-01, apan Two tropinone reductases (TRS) catalyze opposite stereospecific reducti ons at a branching point in the biosynthetic pathway of tropane alkaloids. The two Rs, TR-I and TR-11, reduce the 3-keto group of the common substrate tropinone stereospecifically to a- nd 3p-hy- droxy groups, to produce the stereoisomeric alkamines tropine and pseudotropine, respectively. Sixteen chi- meric TR enzymes were expressed in Escherichia coli, and their stereospecificities, substrate specificities, and K , values for tropinone were compared with those of the wild-type nzymes. tereospecificity nd ubstrate specificity of the chimeric enzymes were closely corre- lated, and the carboxyl-terminal peptides of about 120 amino acid residues, in which 3 residues were different between TR-I and TR-11, were shown to determine both sp eci fici ties . Further diss ection of these peptide s eg- ments resulted in either enzymes with both R ctivities or inactive enzymes. The substrate binding affinity of many chimeric enzymes was much lower than that of wild-type enzymes. These results ndicate hat he stereospecificity of TR is determined by the orientati on of tropinone at the substrate-binding site, which s com- posed mainly of the carboxyl-terminal half region, and also that the amino-terminal half region constitutes the NADPH-binding site as postulated for short chain on- metal dehydrogenases. Tw o tropinone reductases TRs)’ constitute a branching point i n the biosynthesis of tropane alkaloids that are produced in several plant genera belonging mainly to the Solanaceae (1) (Fig. 1). TR-I (E C 1.1.1.206) catalyzes the NADPH-dependent reduction of th e 3-keto group of tropinone to the 3a-hydroxy group of tropine 3a-hydroxytropane), whereas TR-I1 (E C 1.1.1.236) converts the same keto group to the 3P-hydroxy of pseudotropine (+-tropine, 3P-hydroxytropane). Varying ratios of the two TR activities in th e tropane alkaloid-producing spe- * This work was supported in pa rt by a Ja pa n Societ y for the Promo- tion o f Science fellowsh ip for apanese junior scientists ( t o K. N.). The costs o f publica tion o f this a rticle were defra yed in part by the paymen t o f page charges. This article must t herefore be hereby marked “aduer - tisement” in accordance with 18 U.S .C. Section 1734 sol ely to indicate this fact. $ To whom correspondence should be address ed. Tel.: 81-75-753-6382; ‘The abbreviations used are: TR, tropinone reductase; $-tropine, pseudotropine; SDH, short chain non-metal dehydrogenase; G LC, gas- liquid chromatography; PAG E, polyacrylamide gel electro phores is. Fax: 1-75-753-6398. cies (2 ) may determine the metabolite flow at the branching point, since no interconversion between tropine and +-tropine has been detected in vivo (3). The two TRs in Hyoscyamus niger showed both similar and different properties (2). Both Rs bound NADPH with similar high affinity and transferred the pro-S hydrogen of NADPH to tropinone, whereas th e two reductases possess considerably different K,,, values for tropinone and substrate specificities toward variou s cycl ic ketones. Subsequent isolation of th e cDNAs encoding the two TRs from Datura stramonium has revealed that the subunits of TR-I and TR-I1 consist of 27 3 and 260 amino acid residues, respectively, a nd that they share 167 (64%) identical residues. The amino-terminal halves showed higher omology (72% den- tical) than the carboxyl-terminal halves (57%) 4) (Fig. 2). The amino acid sequences of th e TRs also indicated that th e en- zymes belong o th e short chain non-metal dehydrogenase (SDH) family (5). ased on the predicted secondary structures of th e 20 SDHs reported so far, it has been postulated that t h e amino-terminal halves of the SDHs constitute the cofactor- binding sites with alternating a l p structures and ha t the car- boxyl-terminal halves may participate n substrate binding (5). The determination of the three-dimensional structure of an SDH, 3a,20P-hydroxysteroid dehydrogenase (61, essentially substantiated this model. Accordingly, the stereospecificity of the two TR s ma y be de- termined by the orientation f tropinone at th e substrate-bind- ing site, which is presumably located t the carboxyl-terminal half of the enzyme . n this report, we describe he construction of chimeric enzymes between TR-I and TR-11, and we discuss the contribution of the substrate-binding region n determining th e stereospecificity of the two TR reactions. EXPERIMENTAL PROCEDURES Plasmids for Enzyme Expression in Escherichia co li -D NA manipu- lations were performed a s previously descr ibed (7). For expression of wild-type TRs, pTRlEN and pTR2EN were first constructed fr om the D. stramon ium cDN A clo nes pDTRl and pDTR 2 (4), respectively, as fol- lows. A n Nco I site was introduced at the translation start site o f re- spective TR cD NA cl on es by polymerase chain reactio n-media ted site- directed mutagenesis (8) using the mutagenesis primers (5’”- CCATGGAAGAATCAAAAGTGTCCA-3’ or TR-I, and 5’-GAGCCATG- GCTG GAA GGT GG- 3’ for TR- 11; the underli nes denote NcoI si tes ) and the T7 or SP6 primers. Since intrinsic NcoI sites exist in both TR cDNAs, only the 5’- part s o f th e amplified cDNA s (26 0 base pairs for TR-I and 109 base p airs forTR-11) wer e first cloned into the vector pTV119N (Takara Shuzo Co., Kyoto, Japan) with NcoI and another restriction enzyme (Hind111 r AccI). pTV119N is identical to pUC119, exce pt that an Nco I site is introduced at the translati on tart site of the LacZ coding region. nserti on of the remain ing cD NA part s from pDTRl and pDTR2 into the 3”regions of the above plasmids gave pTRlEN and pTR2EN. DNA sequencing confirmed that no unintended base substi- tution h ad been incorpo rated. E. coli strain NM52 2 was transforme d with either pTRlEN or pTR2EN , and the transf ormants were used to establish the expression conditi ons describe d below. To obtain exp ression plasmids for chimeric T R enzymes, f ive restric- tion sites (XbaI, SpeI, BstBI, MluI, and PstI) were introduced by the site-directed mutagenesi s described above nto the TR cod ing regions o f pTRlEN and pT R2EN withou t changi ng the amino acid sequences. The zY”CGAAGCAGCTTATCA’l”ATCT-3’; where Y denotes T o r C), MT 4 (5’-GACAACATACGCGTCAAT-3’), nd MT5 (5’”ITCCCTG- @@Tl“IT ATAT-3’); orresponding antisense primers MTl A-MT5A an d two M13 uni versal primers, RV-N (5”T GTGG AATT GTGA GCGG- 3’, reverse) and M4 (5’-G’R”CCCAGTCACGAC-3’, orward). Six GAAAT-3’), MT 2 (~“CTCAATATACTAGTGLL~TAATGC-~’), T3 (5’- 11695
Transcript
8/3/2019 Keiji Nakajima et al- Opposite Stereospecificity of Two Tropinone Reductases Is Conferred by the Substrate-binding …
From the Department of Agricultural Chemistry,Faculty of Agriculture, Kyoto University,Kyoto 606-01, apan
Two tropinone reductases (TRS) catalyze oppositestereospecific reductions at a branching point in thebiosynthetic pathway of tropane alkaloids. The two Rs,TR-I and TR-11, reduce the 3-keto group of the commonsubstrate tropinone stereospecifically to a- nd 3p-hy-droxy groups, to produce the stereoisomeric alkaminestropine and pseudotropine, respectively. Sixteen chi-meric TR enzymes were expressed in Escherichia coli,and their stereospecificities, substrate specificities, andK, values for tropinone were compared with those of thewild-type nzymes. tereospecificity nd ubstratespecificity of the chimeric enzymes were closely corre-lated, and the carboxyl-terminal peptides of about 120amino acid residues, in which 3 residues were differentbetween TR-I and TR-11, were shown to determine bothspecificities . Further dissection of these peptide seg-ments resulted in either enzymes with both R ctivitiesor inactive enzymes. The substrate binding affinity ofmany chimeric enzymes was much lower than that ofwild-type enzymes. These results ndicate hat hestereospecificity of TR is determined by the orientationof tropinone at the substrate-binding site, which s com-posed mainly of the carboxyl-terminal half region, andalso that the amino-terminal half region constitutes theNADPH-binding site as postulated for short chain on-metal dehydrogenases.
Tw o tropinone reductases TRs)’ constitute a branching pointi n the biosynthesis of tropane alkaloids that are produced inseveral plant genera belonging mainly to the Solanaceae (1 )(Fig. 1). TR-I (EC 1.1.1.206) catalyzes the NADPH-dependentreduction of th e 3-keto group of tropinone to the 3a-hydroxygroup of tropine 3a-hydroxytropane), whereas TR-I1 (E C1.1.1.236) converts the same keto group to the 3P-hydroxy ofpseudotropine (+-tropine, 3P-hydroxytropane). Varying ratiosof the two TR activities in the tropane alkaloid-producing spe-
*This work was supported in part by a Japan Society for the Promo-tion of Science fellowship for apanese junior scientists (to K. N.). Thecosts of publica tion of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “aduer-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
$ To whom correspondence should be addressed. Tel.: 81-75-753-6382;
‘The abbreviations used are: TR, tropinone reductase; $-tropine,pseudotropine; SDH, short chain non-metal dehydrogenase; GLC, gas-
liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
Fax: 1-75-753-6398.
cies (2 ) may determine the metabolite flow at the branchingpoint, since no interconversion between tropine and +-tropinehas been detected in vivo (3).
The two TRs in Hyoscyamus niger showed both similar anddifferent properties (2). Both Rs bound NADPH with similarhigh affinity and transferred the pro-S hydrogen of NADPH totropinone, whereas the two reductases possess considerablydifferent K,,, values for tropinone and substrate specificitiestoward variou s cyclic ketones.
Subsequent isolation of the cDNAs encoding the two TRsfrom Datura stramonium has revealed that the subunits ofTR-I and TR-I1 consist of 27 3 and 260 amino acid residues,respectively, a nd that they share 167 (64%) identical residues.The amino-terminal halves showed higher omology (72% den-tical) than the carboxyl-terminal halves (57%) 4) (Fig. 2). Theamino acid sequences of the TRs also indicated that the en-zymes belong o the short chain non-metal dehydrogenase(SDH) family (5). ased on the predicted secondary structuresof th e 20 SDHs reported so far, it has been postulated that t heamino-terminal halves of the SDHs constitu te the cofactor-binding sites with alternating alp structures and hat the car-boxyl-terminal halves may participate n substrate binding (5).The determination of the three-dimensional structure of anSDH, 3a,20P-hydroxysteroid dehydrogen ase (61, essentia llysubstantiated this model.
Accordingly, the stereospecificity of the two TR s ma y be de-termined by the orientation f tropinone at th e substrate-bind-ing site, which is presumably located t the carboxyl-terminalhalf of the enzyme . n this report, we describe he constructionof chimeric enzymes between TR-I and TR-11, and we discussthe contribution of the substrate-binding region n determiningth e stereospecificity of the two TR reactions.
EXPERIMENTAL PROCEDURES
Plasmids for Enzyme Expression in Escherichia coli-DNA manipu-lations were performed a s previously described (7). For expression ofwild-type TRs, pTRlEN and pTR2EN were first constructed from the D.
stramonium cDNA clones pDTRl and pDTR2 (4), respectively, as fol-lows. A n NcoI site was introduced at the translation start site of re-spective TR cDNA clones by polymerase chain reaction-mediated site-directed mutagenesis (8) using the mutagenesis primers (5’”-CCATGGAAGAATCAAAAGTGTCCA-3’or TR-I, and 5’-GAGCCATG-GCTGGAAGGTGG-3’ for TR-11; the underlines denote NcoI sites) andthe T7 or SP6 primers. Since intrinsic NcoI sites exist in both TRcDNAs, only the 5’-parts of the amplified cDNAs (260 base pairs forTR-I and 109 base pairs for TR-11) were first cloned into the vectorpTV119N (Takara Shuzo Co., Kyoto, Japan) with NcoI and anotherrestriction enzyme (Hind111 r AccI). pTV119N is identical to pUC119,
except that an NcoI site is introduced at the translation tar t site of theLacZ coding region. nsertion of the remaining cDNA parts from pDTRland pDTR2 into the 3”regions of the above plasmids gave pTRlEN andpTR2EN. DNA sequencing confirmed that no unintended base substi-tution had been incorporated. E. coli strain NM522 was transformedwith either pTRlEN or pTR2EN, and the transformants were used toestablish the expression conditions described below.
To obtain expression plasmids for chimeric TR enzymes, five restric-tion sites (XbaI, SpeI, BstBI, MluI, and PstI) were introduced by thesite-directed mutagenesis described above nto the TR coding regions ofpTRlEN and pTR2EN without changing the amino acid sequences. Theprimers used were ive sense primers, MT1 (5”TATACATGTTm-
zY”CGAAGCAGCTTATCA’l”ATCT-3’; where Y denotes T o r C),MT4 (5’-GACAACATACGCGTCAAT-3’), nd MT5 (5’”ITCCCTG-@@Tl“ITATAT-3’); orresponding antisense primers MTlA-MT5Aand two M13 universal primers, RV-N (5”TGTGGAATTGTGAGCGG-
3’, reverse) and M4 (5’-G’R”CCCAGTCACGAC-3’, orward). Six
GAAAT-3’), MT2 (~“CTCAATATACTAGTGLL~TAATGC-~’), T3 (5’-
11695
8/3/2019 Keiji Nakajima et al- Opposite Stereospecificity of Two Tropinone Reductases Is Conferred by the Substrate-binding …
FIG. 1. Reactions of tropinone reductases (TRS). n the biosyn-thetic pathway of tropane alka loids, TR-I and TR-I1 reduce he commonsubstrate tropinone stereospecifically t o tropine (3a-hydroxytropane)
NADPH as a hydride donor.and +-tropine (3P-hydroxytropane), espectively. Both enzymes equire
T R - I e e s k v s m m n n n e G RW S L k t TA LV S k G I I V E E A g L G A r V Y T 5 0TR-I1a GR Wn L e G c TA LV S SG I k g I V E E L A sL G As V YT8
I
TR-I CSRNeKELde CLeiWReKGl nVEgSV 1 S R t E R d k L M q T VA h v F d G K L 1 0 0T R - I 1 C S RN q KE L nd C L t qW R s KG f k V E a S V & S S R s ER q eL M n T VA n h F h G K L8
II
TR-I N I LV N N A G v V I h K E A K D f Te k D Y n i I M g t N F E A AY H L S q i AyPlLKASqn 50TR-I1 N I LV N N A G i V I y K E A K D y T v e D Y s 1 I M S i N F E A AY H L S v l AhPfLKASer 38
-11 -: -
A l n Q m T k s L A c E WA K D N I RV N s Va 2 0 0A m d Q l Tr c L A f E WA K D N I RV N g V g 18 8
TR-I P G V I l T p LV E t a I k k n P h Q K E e i d n f I v k t prngRaGkPqE v s A l i A F L C F 25 0T R - I 1 P G V I a Ts LV E m t I - q d P e Q K E n l n k l I d r c a l r R m G e P k E 1 a A m v A F L C F 3 1
V
TR-I PA A S Yi T G Q I I w a D G G f t A N gG FTR-I1 PA A S Yv T G Q I I y v D G G l m A N CG F
" -2 1 32 6 0
"11, an d division of the TR polypeptide into six regions. TheFIG. . Comparison of the amino acid sequences of T R - I an d
amino acid sequences of TR-I and TR-I1 rom D. stramonium werealigned by the computer software GeneWorks (IntelliGenetics). The TRpolypeptides were divided nto six regions (I-VI) at five well conserved
in both TRs are shown in uppercase letters, whereas residues that arestretches of amino acid residues. Amino acid esidues that are dentical
th at ar e strictly conserved in SDHs (5) are shown in black boxes.different between the two enzymes are in lowercase letters. Six residues
polymerase chain reactions were performed with each of pTRlEN and
MTlJ"T2A, MT2/MT3A, MT3/MT4A, MT4/MT5A, and MT5/M4. ThepTR2EN as a template with the primer combinations of RV-N/MTlA,
amplified DNA fragments were cloned into pTV119N, pBluescript I1SK- Stratage ne), or pcDNAII (Invitrogen), either by the introducedrestriction sites or by using synthetic linkers. M e r confirmation of thenucleotide sequences, the DNA fragments were successively ligated toeach other at th e introduced restriction sites t o give pTRlMS andpTR2MS. In E. coli st ra in NM522, pTRlMS and pTRZMS, respectively,expressed wild-type TR-I and wild-type TR-I1 nzymes, and theamounts and the kinetics of their express ions were comparable o thoseof pTRlEN and pTR2EN. Plasm ids for chimeric TR enzymes were con-structed by exchanging cDNA segments between pTRlMS an d pTR2MSat t he introduced restriction sites and the EcoRI sites at the 3'-ends ofthe cDNA inserts.
Bacterial Culture, Protein Extraction, and Enzyme Assay-E. coliNM522 harboring the expression plasmids were inoculated in 5 ml ofM9 media supplemented with 0.2% (w/v) -glucose, 0.04% w/v) L-aminoacids, 1 m MgSO,, 1 l l ~ gCI,, 0.001% (w/v) hiamine, and 200 p g / dampicillin, and incubated a t 37 "C with agitation (300 rpm). When th eA, of the culture reached -0.5, isopropyl-1-thio-P-D-galactopyranosidewas added at a final concentration of 1 m, and the incubation wascontinued for an additional 16 h. The bacteria were harvested by cen-trifuga tion, washed with STE (10 ris-HC1, pH 7.5,l m EDTA, 150II~M NaCl), and lysed by sonication on ice in 100 pl of the extractionbuffer (0.1 M potass ium phosphate, pH 7.0, 3 m dithiothreitol). m e rcentrifugation (10,000 x g ) or 15 min at 4 "C, 1-5 p1 of the supernatant(soluble fraction) was directly added to the TR reaction mixture (1 ml).Due to its accuracy in measuring the initial reaction rate, the spectro-photometric method 2) was preferent ially used for TR assay in kineticstudies and analysis of substrate specificity, while gas-liquid chroma-tography (GLC) (2) was used to measure low TR activities in several
A enzyme type TR-I TR-III
TR12-2
TR12-3 && .$i
TR12-4 :&. ,
TR12-5 m
.. ..~,,. >>, , >><<..... .,.:.,
wild-typeTR-I -C TR121-35 . . ,. .
ND.TRl21-34NDD
ND
, ,~.~.><I , I .
. . ,
..,.,
TR212-34
L
105 103 103 l o 5
( pKat I rng TR protein )
FIG. 3. R ctivities of chimeric TR nzymes. Specific activities ofeach recombinant enzyme were calculated as described in th e text. TRactivities are expressed in a logarithmic scale with the detection limit(10 picokataldmg TR protein) located at the origin. The structure ofeach enzyme subunit i s shown by dark gray and light gray boxes, whichrepresent the TR-I and TR-I1 polypeptides, respectively. ND, not de-tected.
chimeric enzymes and to dete rmine stereospecificity. Neithe r of thesetwo assay m ethods detected any background TR activities in crudeenzyme preparations from bacteria th at had been transformed with theempty vector when ropinone, 3-quinuclidinone, and N-propyl-4-piperi-done were used s substrates. The total amount of protein in the extractwas assayed as previously described (9). The precipitate of the finalcentrifugation was solubilized n 100 m ithiothreitol, 2% (w/v) SDS t100 "C an d used as the insoluble fraction. The protein extracts wereanalyzed by SDS-PAGE, and the Coomassie Brilliant Blue-s tained gelswere used to est imat e the amounts of expressed proteins by densitom-etry (GelScan XL , Pharmacia LK B Biotechnology Inc.). For chimeric
enzymes with low TR activit ies, bacteria were grown in a large scaleculture (100 ml), and the protein extracts were desalted by gel-filtrationcolumns (NAP-10, Pharmacia). Up to 200 pl of the elua te was used forthe TR assay.
RESULTS AND DISCUSSION
Construction of Chimeric TR Enzymes-We fir st performed alimited proteolysis of the purified TR enzymes to locate flexibleregions tha t ar e xposed to th e solvent. Such loop regions maybe used as favorable sites for exchanging TR polypeptideswhen constructing chimeric enzymes. Under mild conditions(TR:protease = 1OOO:l or 100:1, ncubation at 15 or 25 "C), othtrypsin and V8 protease failed to digest the TR enzymes. Ad-dition of a larger amount of the pro teases TR:protease = 20:l)at an elevated incubation temperature (37 "C) esulted in thepar tia l hydrolysis of TR enzymes into smal l peptide fragme nts(data not hown). These esults indica te t hat n either f the TR
8/3/2019 Keiji Nakajima et al- Opposite Stereospecificity of Two Tropinone Reductases Is Conferred by the Substrate-binding …
Stereospecificity in Chimericopinoneeductases 11697
I TR12-1 I TR21-1
I TR12-2 I TR21-2
I TR12-3 I TR21-3123 .
I ~TR12-4 I TR21-4
I TR12-5 I TR21-5
I wild-typeTR-I Iwild-typeTR-II
100%ubstrate I 100%
relative activityFIG. . Substr ate specificities of chimeric TR enzymes. The ac-
tivities with 3-quinuclidinone (bar 2; 0 ) nd N-propyl-4-piperidone ba r3 ; Dl are expressed as percentages of the activity with tropinone (bar 1 ;B) for each enzyme. N D , not detected.
enzymes contain loop-like stru ctu res that are accessible by aprotease molecule.
We next divided each TR polypeptide into six regions (regionsI-VI in Fig. 2) at relatively long stretches of identical aminoacid residues. T he chimeric TR enzymes were constructed byexchanging these six regions between TR-I and TR-11. SDS-PAGE analysis of the E . coli extracts detected the expressedchimeric proteins mostly in the soluble fractions, as observedfor wild-type TRs (data not shown), but the amoun ts of therecombinant proteins varied considerably among the chimericenzymes.
Stereospecificity-TR activit ies in the soluble fractions wereassayed by GLC (Fig. 3). The proportion of a TR protein to thetotal soluble protein was estimated by densitometric scanningof a Coomassie Brill iant Blue-stained gel after SDS-PAGE, andthe val ue (5-14%) was used to calculate the TR activity to
adjust the different expression levels of chimeric enzymes. Asshown in Fig. 3 A , chimeric enzymes containing region IV of
TABLEK,,,values of wild-type and chimeric TR enzymes fo r tropinone
Enzyme activities were measured or more than five concentrations oftropinone. For the chimeric enzymes with both TR act ivities, the sum ofthe two activities was used for calculation . The K , values ( * standard
method (12).deviation) were determined by using Wilkinson’s statistical analysis
TR-I1 (TR12-1, TR12-2, and TR12-3) showed higher TR-I1 ac-tivity th an TR-I activity, whereas the chime ras ontaining thecorresponding region of TR-I (TR12-4 and TR12-5) had higherTR-I activity than TR-I1 activity, although the differences be-tween the two TR activities were not very large in the chim eraTR12-4 (TR-1:TR-I1 = 1 O : l ) . Simil ar re sults were obtained forthe chimeras in which combinations of the fused polypeptideswere reversed (Fig. 3B), although in this series the chimeraTR21-4 did not exhibit either TR activity.
Next, six additional chimeric enzymes in which only one orboth of regions N and V substituted were expressed in E . coli(Fig. 3C). R activities and stereoselectivities of these chimeraswere severely impaired; two (TR121-35 and TR121-34) wereinactive, while three (TR121-45, TR212-34, and TR212-45)
showed the two TR activities at low, but comparable, levels.TR212-35 showed only TR-I activity a t a low level. Since mul-timeric assembly is required for activity of SDHs (lo), he qua-ternary structures of TR21-4, TR121-35, TR121-34, and wild-type TRs were examined by gel-filtration chromatography.Native molecular masses of these three chimera (67-75 kDa)were comparable to those of wild-type TR-I (79 kDa) and TR-I1(82 kDa), indicating correct su bunit assembly. These resu ltssuggest t ha t both regions IV an d V of TR-I and TR-I1 a c t ad-ditively to deter mine he stereospecificity of these two en-zymes.
Substr ate Binding-The two TRs have different substr atepreferences for cyclic ketones. For example, TR-I of H. igereffectively reduces -quinuclidinone and 8-thiabicyclo-[3,2,lloctane-3-one, whereas TR-I1 of the sam e pla nt showslittl e activity toward these keton es 2). Conversely, piperidonesare good substrates for TR-I1 but poor substrates for TR-I (2).3-Quinuclidinone and N-propyl-4-piperidone were used to testsubstrate preferences of the chimeric enzymes (Fig. 4). Wild-type TR enzymes of D . stramonium showed the same subst ratespecificity toward these cyclic ketones as the enzymes of H.niger. Wild-type TR-I efficiently reduced 3-quinuclidinone,while very little activity was detected for N-propyl-4-piperi-done. A simi lar substr ate preference was observed for the chi-mer as TR21-1, TR21-2, TR21-3, and TR12-5. Wild-type TR-I1reduced only N-propyl-4-piperidone, and essentially the samesubstrate specificity was found for thechimeras TR12-1,TR12-2, TR12-3, and TR21-5. The chimera TR12-4 showed ahigh activity with both substrates , wher eas TR21-4, which ha dno activity toward tropinone, reduced eithe r of these ketones.The substr ate reference of these chimeric enzymes was losely
8/3/2019 Keiji Nakajima et al- Opposite Stereospecificity of Two Tropinone Reductases Is Conferred by the Substrate-binding …
correlated to their stereospecificity; chimeras that exhibited ahigh TR-VlX-11 activity rat io showed TR-I-type subs tra tespecificity, whereas chime ras that exhibited a low TR-VTR-I1ratio showed TR-11-type. These result s indicate th at regions IVan d V dete rmine both th e stereospecificity of tropinone reduc-tion and the substra te pecificity, and together contain most ofthe substrate-binding ites.
Although the two TR s bind th e common substrate tropinone,
the ir affinity oward ropinone differs considerably. For theTRa of both H . niger (2) and D. tramonium (111, TR-I1 showslower K , valu es than TR-I. Chimeric enzymes were also ana-lyzed for K , values (Table I). Th e affinity of most chimericenzymes toward tropinone was significantly lower th an th at ofthe wild-type TRa, except for TR12-1 and TR12-2, whichshowed low K,,, alues comparable o those etermined for wild-type TR-11. Thes e res ul ts are consistent wi th he proposedthree-dimensional structure for the SDH, 3a,2OP-hydroxy-steroid dehydrogenase, in which a few amino acid residues atthe amino-terminal half also parti cipate in building up thesteroid-binding site (6).
Concluding Remarks-The bacte rial 3a,20P-hydroxysteroiddehydrogenase, the sole SDH whose x-ray stru ctur e has been
elucidated at a resolution of 2.6 A (61, oxidizes either the 301- or2Op-hydroxyl groups of androstane and pregnane derivatives.This dehydrogenase possesses only one cofactor- and one sub-strate-binding site per subunit, indicating that the egio-speci-ficity of the steroid dehydrogenation e sul ts from the binding ofthe steroid in two orientat ions near the same cofactor at th esame catalytic site 6). ikewise, the ability of several chimericTRs to reduce the 3-keto group nto two opposite directions canbe explained by the binding of tropinone in two orientations at
the same catalytic site. Stric t stereospecificity would emerge asone of the two orientations becomes favored by modifications toth e tropinone-binding site. For example, alteration of at most25 amino acid resi dues in region IV is sufficient to convertchimera TR212-34, which ha s both TR activities, i nto hehighly stereospecific wild-type TR-11, whereas str ict TR-I speci-ficity can be obtained by introducing a maximum of 28 aminoacid substitutions into egion V of TR212-34 to produce TR212-
35. Similar changes may have occurred during evolution ofTRs; aft er gene duplication, an ancest ral TR enzyme evolvedinto two TRs with opposite stereospecificities by accumulatingmutations at the tropinone-binding site that is mainly com-prised of regions IV an d V.
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