Báo cáo khoa học: to 3a-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 using fluorescence stopped-flow procedures pptx

Transient-phase kinetic stud-ies using the fluorescence stopped-flow method were con-ducted with 3a-HSD to characterize the nucleotide binding mechanism.. The binding of oxidized nucleotid

Transient-phase kinetic studies on the nucleotide binding

B-0831 using fluorescence stopped-flow procedures

Shigeru Ueda1, Masayuki Oda2, Shigeyuki Imamura1and Masatake Ohnishi2

1

Department Diagnostics Research and Development, Division of Fine Chemicals and Diagnostics, Asahi Kasei Pharma Corporation, Shizuoka, Japan;2Department of Cellular Macromolecule Chemistry, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto, Japan

The dual nucleotide cofactor-specific enzyme,

3a-hydroxy-steroid dehydrogenase (3a-HSD)from Pseudomonas sp

B-0831, is a member of the short-chain dehydrogenase/

reductase (SDR)superfamily Transient-phase kinetic

stud-ies using the fluorescence stopped-flow method were

con-ducted with 3a-HSD to characterize the nucleotide binding

mechanism The binding of oxidized nucleotides, NAD+,

NADP+ and nicotinic acid adenine dinucleotide

(NAAD+), agreed well with a one-step mechanism, while

that of reduced nucleotide, NADH, showed a two-step

mechanism This difference draws attention to previous

characteristic findings on rat liver 3a-HSD, which is a

member of the aldo-keto reductase (AKR)superfamily

Although functionally similar, AKRs are structurally

dif-ferent from SDRs The dissociation rate constants

associated with the enzyme–nucleotide complex formation were larger than the kcat values for either oxidation or reduction of substrates, indicating that the release of cofac-tors is not rate-limiting overall It should also be noted that

kcatfor a substrate, cholic acid, with NADP+was only 6%

of that with NAD+, and no catalytic activity was detectable with NAAD+, despite the similar binding affinities of nucleotides These results suggest that a certain type of nucleotide can modulate nucleotide-binding mode and fur-ther the catalytic function of the enzyme

Keywords: aldo-keto reductase superfamily; nucleotide-binding; fluorescence stopped-flow method; 3a-hydroxyster-oid dehydrogenase; short-chain dehydrogenase/reductase superfamily

The NAD(P)+-dependent enzyme, 3a-hydroxysteroid

dehydrogenase (3a-HSD), catalyzes the reversible

intercon-version of hydroxy and oxo groups at position 3 of the

steroid nucleus, and has been found in many mammalian

cells and microorganisms [1,2] Prokaryotic 3a-HSDs have

been described in Eubacterium lentum [3], Clostridium

perfringens[4], Pseudomonas putida [5], Comamonas

(Pseu-domonas) testosteroni ATCC11996 [6–9], and Pseudomonas

sp B-0831 [10] Despite similar substrate specificities,

eucaryotic and prokaryotic 3a-HSDs belong to two

differ-ent protein superfamilies: viz, the eucaryotic and

prokary-otic enzymes, respectively, belonging to the aldo-keto

reductase (AKR)(EC 1.1.1.213)and the short-chain

dehy-drogenase/reductase (SDR)superfamilies (EC 1.1.1.50) [11–14] Although structurally different, both HSDs are functionally similar; the AKRs are monomeric and have an a/b-barrel fold, whereas the SDRs are dimeric or tetrameric and contain a Rossmann nucleotide-binding fold

We have previously cloned 3a-HSD from Pseudomonas

sp strain B-0831 and expressed it in Escherichia coli [10] The SDS/PAGE and gel-filtration analyses revealed that this enzyme forms a homodimer comprised of two 25 kDa monomer proteins The amino acid sequence shares about 50% identity with that of Comamonas (Pseudomonas) testosteroni ATCC11996 [15], and contains two motifs common to SDRs, the Gly-X-X-X-Gly-X-Gly cofactor-binding and Tyr-X-X-X-Lys substrate-cofactor-binding motifs Because 3a-HSD from C testosteroni can catalyze not only the oxidoreduction at position 3 of the steroid nucleus but also carbonyl reduction of a variety of nonsteroidal aldehydes and ketones, the enzyme was named as 3a-hydroxysteroid dehydrogenase/carbonyl reductase (3a-HSD/CR)[15,16] In a similar manner,

P.sp B-0831-derived 3a-HSD also shows carbonyl reduc-tase activity [17] Different from the NAD+-dependent 3a-HSD/CR, P sp B-0831-derived 3a-HSD has the ability to use not only NAD+ but also NADP+ In clinical diagnostics, the enzyme has been used for the measurement of total bile acids in serum [18] Further-more, a highly sensitive and unique enzyme cycling method for total bile acids assay has been developed

Correspondence to M Oda, Graduate School of Agriculture,

Kyoto Prefectural University, 1–5, Shimogamo Nakaragi-cho,

Sakyo-ku, Kyoto 606–8522, Japan.

Fax: + 81 75 7035673, Tel.: + 81 75 703 5673,

E-mail: oda@kpu.ac.jp

Abbreviations: 3a-HSD, 3a-hydroxysteroid dehydrogenase; AKR,

aldo-keto reductase superfamily; CR, carbonyl reductase; NAAD + ,

nicotinic acid adenine dinucleotide; SDR, short-chain dehydrogenase/

reductase superfamily.

Enzyme: 3a-hydroxysteroid dehydrogenase (EC 1.1.1.213,

EC 1.1.1.50).

(Received 3 February 2004, revised 5 March 2004,

accepted 15 March 2004)

using this B-0831 enzyme in the presence of excessive

thio-NAD+ (NAD+ analogue)and NADH, which are

commercially available reagents [19]

Rat liver 3a-HSD, the first HSD to be assigned to the

AKRs, is involved in bile acid biosynthesis, and participates

in the inactivation of circulating steroid hormones such as

androgens, progestins and glucocorticoids [20] Its

nucleo-tide cofactor-binding mechanisms have been extensively

studied [21–27] This enzyme shows NADP+ preference

and follows an ordered bi-bi reaction mechanism with

nucleotide binding first The binding kinetics of NADP(H)

by the fluorescence stopped-flow method showed a two-step

mechanism which consists of fast formation of a loose

complex followed by slow formation of a tightly bound

complex [26], and the dissociation of NADP(H)is not

rate-limiting The latter finding discriminates the rat enzyme

from other AKRs, such as human and pig muscle aldose

reductases [28,29]

As well as rat liver enzyme, 3a-HSD/CR from C

testo-steroni ATCC11996 follows an ordered bi-bi mechanism

with pyridine nucleotide binding first and dissociation last [7]

In contrast to 3a-HSDs in AKRs, however, little is known

about the nucleotide-binding mechanism of 3a-HSD in

SDRs In terms of nucleotide binding to the enzyme, the

nicotinamide ring adopts syn conformation in SDRs while it

adopts anti conformation in AKRs, exhibiting the opposite

stereospecificity of hydride transfer [27] Therefore, it is of

great interest to elucidate the mechanism of the nucleotide

binding to 3a-HSD from P sp B-0831 as a member of SDRs

In the present study, we analysed transient-phase kinetics

of nucleotide binding to bacteria-derived 3a-HSD by

fluorescence stopped-flow measurements for the first time

Transient-phase kinetics can distinguish between the

for-mation and decay of individual complexes on either one- or

multiple-step reaction pathways Application of this method

to 3a-HSD revealed that the reaction mechanism depends

on the type of nucleotides, and is different from that of

rat liver 3a-HSD In addition to the unique features of

the kinetic mechanism, nucleotide preference of this enzyme

could be elucidated in comparison with the functional and

structural information of other SDRs and AKRs, such as

C testosteroni3a-HSD/CR and rat liver 3a-HSD

Materials and methods

Materials

NAD+, NADP+, NAAD+, NADH, and NADPH

(Ori-ental Yeast Co., Ltd in Japan), steroids (Sigma Chem Co.)

and other chemicals were of the highest commercial quality

available Recombinant 3a-HSD from Pseudomonas sp

strain B-0831 was expressed in E coli and purified as

described previously [10] The protein purity was

deter-mined to be over 95% by SDS/PAGE analysis, and the

protein concentration was determined by the method of

Lowry et al [30]

Steady-state kinetic study

Assays were carried out at 37C or 15 C in 40 mMTris/

HCl (pH 8.5), in the presence of 1 mMNAD(P)+, 0.025%

nitrotetrazolium blue (NTB), 0.4% Triton X-100, and 2.5

units/mL diaphorase (Asahi Kasei)for oxidation reaction, and in the presence of 0.3 mM NAD(P)H for reduction reaction [10] In the assay for substrate specificity, various substrate concentrations were used for the determination of

Km and kcat To determine Km for the NAD(P)+ and NAD(P)H binding, 1 mMcholic acid and 0.8 mM dehydro-cholic acid were used, respectively The reaction was initiated by adding the enzyme, and terminated by adding

2 mL of 0.5% SDS after incubation for 5 min The Kmand

kcat values for oxidation reaction were determined by measuring the formation of formazan dye at 550 nm, and those for reduction reaction were determined by measuring the decrease of NAD(P)H at 340 nm, using a Shimadzu UV-2200 spectrophotometer In the inhibition study by NAAD+, 1.0 mMcholic acid (substrate)was used in the presence of NAD+with concentration ranging from 5.0 lM

to 200 lM, and 2.0 mMNAAD+as an inhibitor The Km

and Kivalues were calculated by the nonlinear least squares method using the Taylor expansion [31]

Fluorescence titration measurements The nucleotide binding to 3a-HSD was measured by monitoring the quenching of intrinsic enzyme fluorescence upon incremental addition of nucleotides Emission spectra (300–500 nm)were recorded on a JASCO Corporation FP-777 fluorescence spectrophotometer at 280-nm excita-tion All titrations were carried out in 3-mL volumes with 0.49 lM of the enzyme in 50 mM Tris/HCl (pH 8.5)at

15C The total volume change due to the addition of nucleotide was less than 2% The Kdvalue was determined

by the nonlinear least squares method [31]

Stopped-flow kinetic study The time course of fluorescence intensity caused by nucleo-tide binding to 3a-HSD was monitored through cut-off filters (Toshiba Kasei Kogyo), UV-31 with 50% transmittance

at 310 nm for oxidized nucleotides and UV-42 with 50% transmittance at 420 nm for reduced nucleotides, equipped with an Otsuka Electronics RA-401 stopped-flow apparatus

at 280-nm excitation by a 200 W D2lamp at 15C [32] Using

a quartz cell (inner diameter: 2 mm), the dead time of the apparatus was determined as 1.3 ms under the experimental conditions described in a previous study [33] In order to determine the reliable kinetic range in the present experi-ments, we measured the reduction rate of 2,6-dichloro-phenol-indophenol by ascorbate, and confirmed the linearity

of the apparent first-order rate constant, kapp, within the range of 0–400 s)1in the Guggenheim plot [34]

Various concentrations of nucleotide solutions and 3a-HSD in 50 mMTris/HCl (pH 8.5)were introduced into the stopped-flow apparatus by means of a separate syringe The kapp value for each nucleotide concentration was determined in triplicate

Data analysis of rapid reaction The apparent first-order rate constant, kapp, was determined from the Guggenheim plot When the plots followed the linear dependence of kapp against initial concentration of nucleotide, Eqn (1)was adopted:

FEBS 2004 Transient kinetics of nucleotide binding to 3a-HSD (Eur J Biochem 271)1775

Eþ Nuc !

k þ1



k 1

where E is the enzyme, Nuc is the nucleotide, E-Nuc is the

enzyme–nucleotide complex, and k+1, k)1are the

transient-phase kinetic rate constants On the condition that the initial

concentration of nucleotide, [Nuc], is greater than E, kapp

is thus expressed as Eqn (2)[35]:

kapp¼ kþ1½Nuc þ k1 ð2Þ The values of k+1and k)1were determined from the slope

and the intercept of the plot of [Nuc] vs kapp, respectively

The dissociation constant of E-Nuc, K)1, can be calculated

from Eqn (3):

In contrast, when the plots of [Nuc] vs kappwere nonlinear,

Eqn (4)was applied:

Eþ Nuc !

k þ1



k 1

E-Nuc !

k þ2



k 2

where E-Nuc is an intermediate (loosely bound)form and

E*-Nuc is a more tightly bound isomerized form, and k+1,

k)1, k+2 and k)2 are the transient-phase kinetic rate

constants In this mechanism, the reciprocal of the slower

relaxation time, kapp, is expressed as Eqn (5)when

[Nuc] >> [E] [36]

kapp¼ kþ2[NADH]

K1þ [NADH]þ k2 ð5Þ where K)1(¼ k)1/k+1)is the dissociation constant for the

intermediate E-Nuc complex The k)2value can be

estima-ted by linear extrapolation in the low [Nuc] region of kappvs

[Nuc] plot The k+2and K)1 values were obtained with

the k)2value determined above, by the reciprocal of the

intercept and the slope/intercept of the secondary plot of

1/(kapp) k)2)vs 1/[Nuc], respectively [35] In the two-step

mechanism, the overall dissociation constant, Kd, for the

E-Nuc complex can be calculated from Eqn (6)

Kd¼ K1=½1 þ ðkþ2=k2Þ ð6Þ

Results

Substrate specificity

The Km and kcat values of recombinant 3a-HSD from

P.sp B-0831 toward typical substrates for both forward

and reverse reactions were determined (Table 1) There

was no difference between native and recombinant

enzymes with respect to these kinetic values (data not

shown) Compared with the enzyme from C testosteroni

ATCC11996, the Kmvalue for androsterone was 10 times

higher (210 to 31.1 lM), while that for

5a-androstan-3,17-dione in the reverse reaction was of the same magnitude

(44 to 42.2 lM)[15] For cholic acid as a substrate, the

kcat value with NADP+ was only 6% of that with

NAD+, whereas both of the Km values were similar In

the case of dehydrocholic acid as a substrate, the kcat

value with NADPH was 4.2% of that with NADH

whereas the K value with NADPH was of the same

order in magnitude as that with NADH The kcat values

at 15C were also measured for comparison with the dissociation constants of nucleotide binding obtained by fluorescence stopped-flow procedure, as described below Nicotinic acid adenine dinucleotide (NAAD+), a precur-sor of NAD+, does not work as cofactor in the reaction [37] However, the oxidation reaction of cholic acid in the presence of NAD+was inhibited by NAAD+with 373 lM

of the inhibitor constant (Ki)in a competitive manner, indicating that NAAD+ also binds to the enzyme The NAAD+binding to the enzyme, together with the binding

of other nucleotides, was also investigated by both fluores-cence titration and stopped-flow measurements, as des-cribed below

Fluorescence titration with nucleotides

P.sp B-0831-derived 3a-HSD irradiated an intrinsic fluor-escence emission spectrum of 336 nm kmax at 280 nm excitation The incremental addition of NAD+and NADH quenched the fluorescence emission signal (Fig 1) Plots

of the degree of decrease in fluorescence intensity against NAD(P)+, NAAD+, and NAD(P)H examined here were fitted to a saturation absorption isotherm, yielding the Kd value by the nonlinear least squares method The Kdvalues for NAD+and NADP+approximated well, while the Kd value for NADPH was 16-fold larger than that of NADH (Table 2) As a reference, steady-state kinetic assays were performed The Kmvalue of NADP+was slightly larger than that of NAD+, and the Kmvalue of NADPH was 23-fold higher than that of NADH (Table 2)

Transient-phase kinetics on oxidized nucleotide binding

To determine whether nucleotide binding to 3a-HSD from

P.sp B-0831 constituted a one- or two-step mechanism, we used fluorescence stopped-flow measurement The time course for oxidized nucleotide binding demonstrated that binding of not only NAD+but also NADP+is apparent by the fluorescence kinetic transients The kapp values for NAD+ and NADP+ were 84–225 s)1 and 62–129 s)1, respectively From the relationship of kapp against the respective initial concentrations of NAD+ and NADP+

Table 1 Steady-state kinetic parameters for 3a-HSD from Pseudo-monas sp B-0831 withsteroid substrates n.d., Not determined.

a

(l M ) k cat a

(s)1) k cat

b

(s)1)Nucleotide Androsterone 210 ± 8c 134 ± 24c 16.4 NAD+ Cholic acid 31 ± 2 c 75 ± 1 c n.d NAD +

Deoxycholic acid 54 ± 3 89 ± 6 n.d NAD+ Cholic acid 72 ± 2 4.5 ± 0.5 n.d NADP+ 5a-Androstan-3,

17-dione

5a-Androstan-17b-ol-3-one

Dehydrocholic acid 17 ± 1 78 ± 4 9.3 NADH Dehydrocholic acid 85 ± 3 3.3 ± 0.3 n.d NADPH

a Determined at 37 C b Determined at 15 C c Data were taken from Ueda et al [37].

(Fig 2), the linear dependence of kappagainst both [NAD+]

and [NADP+] indicated that the oxidized nucleotide

cofactor binding is in accordance with a simple one-step

mechanism The NAAD+binding also showed a one-step

mechanism (data not shown) The kinetic rate constants,

k+1and k)1, and the dissociation constant, K)1, for the

nucleotide binding to 3a-HSD (Table 3)are of the same order to the corresponding Kmvalue indicated in Table 2, although the measurements were conducted at different temperatures

Fig 1 Fluorescence emission spectra from binary complexes of

3a-HSD withNAD+or NADH, where an intrinsic fluorescence

emis-sion spectrum of 336 nm k max for 3a-HSD was portrayed at 280 nm

excitation (A)Spectra of 6.43 l M 3a-HSD in the absence (solid line)or

presence of 6.7 l M (dotted line), 23.3 l M (broken line), and 90 l M

(dot-dashed line)NAD + (B) Spectra of 0.49 l M 3a-HSD in the

absence (solid line)or presence of 1.3 l M (dotted line), 2.6 l M (broken

line), and 6.7 l M (dot-dashed line)NADH.

Table 2 K d values obtained from fluorescence titration and K m values obtained from steady-state kinetics for eachnucleotide binding.

Nucleotide

Fluorescence titration

K d a

(l M )

Steady-state kinetics

K m b

(l M )

a

Determined at 15 C b

Determined at 37 C c

K i value, deter-mined in the inhibition assay.

Fig 2 Dependence of k app on the initial concentration of NAD + (A) and NADP+(B) All reactions were carried out in 50 m M Tris/HCl (pH 8.5)at 15 C with 0.56 l M enzyme and various concentrations of NAD+(from 2.5 to 75 l M )or NADP+(from 10 to 100 l M ) Values were expressed as the mean ± SD The lines were drawn according to Eqn (2), using k +1 ¼ 1.79, k )1 ¼ 84.8 for NAD + and k +1 ¼ 6.34,

k)1¼ 54.5 for NADP +

FEBS 2004 Transient kinetics of nucleotide binding to 3a-HSD (Eur J Biochem 271)1777

Transient-phase kinetics on reduced nucleotide binding

In the case of reduced nucleotide binding, the increase of

fluorescence intensity in NADH binding could be

monit-ored through a UV-42 cut-off filter The kapp value was

56–145 s)1, and the dependence of kapp on the initial

concentration of NADH showed a hyperbolic curve

(Fig 3) Different from oxidized nucleotides, the kinetic

feature of the NADH binding was consistent with a

two-step mechanism, which involves a fast bimolecular

associ-ation process followed by a slow unimolecular isomerizassoci-ation

process The kinetic rate constants, k+2and k)2, and the

dissociation constants, K)1 and Kd, are summarized in

Table 3

The binding of NADPH to 3a-HSD was also investigated

by stopped-flow measurement Although the quench in

intrinsic enzyme fluorescence emission spectrum following

NADPH binding was observed here as well, there were no

kinetic transients over a wide range of nucleotide

concen-trations These results suggest different binding modes

toward NADPH and NADH

Discussion

The transient-phase kinetics revealed that the binding of

oxidized nucleotide cofactors, NAD+ and NADP+, to

3a-HSD from P sp B-0831 follows a one-step mechanism,

while the binding of reduced nucleotide cofactor, NADH,

follows a two-step mechanism The binding of NAAD+, not a cofactor but a competitive inhibitor, also follows a one-step mechanism, as is the case of the oxidized cofactors The validity of the proposed mechanism was supported by similar dissociation constants between the K)1or Kdvalues determined by stopped-flow measurements and the Kd values determined by the fluorescence titration (Tables 2 and 3) For binding of oxidized nucleotides, the Kdvalues

by fluorescence titration ranged from 2.2- to 3.6-fold the corresponding K)1 values The Kdvalue for the NADH binding obtained by fluorescence titration was 2.5-fold that obtained by stopped-flow analysis

In HSD-related enzymes, whether or not the binding or release of nucleotide cofactors is the rate-limiting step in the reaction remains an issue [26,28,29] The rate constant k)1 for release of NAD+in a one-step mechanism (84.8 s)1) was larger than the kcatvalue (11.6 s)1)for 5a-androstan-3,17-dione reduction (Tables 1 and 3), indicating that the dissociation of NAD+ from the B-0831 3a-HSD is not rate-limiting overall, a finding that coincides well with dissociation reported on the enzyme from rat liver [26] The rate constant k)2for release of NADH from the B-0831 3a-HSD in a two-step reaction (24 s)1)was slightly larger than the kcatvalue for androsterone oxidation (16.4 s)1) While the dissociation of NADH in this reaction could contribute to rate limiting, those in the oxidation of other substrates examined in this study would not be rate limiting

as the kcatvalues are smaller than that for androsterone oxidation (Table 1)

The rapid kinetic transients were not observed in the NAD(H)binding to rat liver 3a-HSD, suggesting different modes of binding toward NAD(H)and NADP(H)[26] The

Kdvalues of NADP(H)binding were much smaller (c., three order in magnitude)than those of NAD(H)[27] It was concluded the interaction between the 2¢-phosphate of NADP+and Arg276 was essential for the observation of kinetic transients In contrast, rapid kinetic transients were observed in the binding of NAD+, NADP+and NADH to

P.sp B-0831-derived 3a-HSD, albeit such was the case in NADPH binding The oxidized nucleotide cofactors NAD+and NADP+bound to the enzyme with similar affinity, following a one-step catalysis In the reduced nucleotide cofactors, the respective binding affinities were different, and the relatively higher binding affinity nucleo-tide, NADH, only showed a two-step catalysis These results indicate that the reaction mechanism depends on the type of nucleotides, and is different from that of rat liver 3a-HSD

In order to discuss the nucleotide cofactor preference, the amino acid sequence in the nucleotide binding region was compared with those of other SDRs Similar to 3a-HSD from P sp B-0831, 3a-HSD/CR from C testosteroni and

Table 3 Transient-phase kinetic parameters for the formation and decay of enzyme-nucleotide complexes.

Nucleotide k +1 ( M )1 Æs)1) k)1(s)1) K)1(l M ) k +2 (s)1) k)2(s)1) K)1(l M ) K d (l M ) NAD+ 1.79 ± 0.1 · 10 6

84.8 ± 0.8 47.6 ± 2.6 NADP+ 6.34 ± 1.0 · 10 5

54.5 ± 3.3 87.2 ± 8.0 NAAD + 3.67 ± 0.3 · 10 5 41.5 ± 1.9 113.1 ± 4.7

Fig 3 Dependence of k app on the initial concentration of NADH All

reactions were carried out in 50 m M Tris/HCl (pH 8.5)at 15 C, with

0.84 l M enzyme and various concentrations of NADH (from 5 to

37.5 l M ) Values were expressed as the mean ± SD The line was drawn

according to Eqn (5), using k +2 ¼ 278, k )2 ¼ 24, K )1 ¼ 39 Insert:

plot of 1/(k app – k)2)vs 1/[NADH] The k)2value was estimated by

linear extrapolation in the low concentration region of NADH.

7a-HSD from E coli have common Asp residues at position

32 (numbering according to the B-0831 3a-HSD), which

is highly conserved among enzymes preferring NAD+

[14,16,38,39] It is noteworthy that the substitution of Asp

for Thr38 in mouse lung CR changed the nucleotide

preference from NADP+ to NAD+ [40] The crystal

structure of C testosteroni-derived 3a-HSD/CR complexed

with NAD+has shown that Ile33, located next to Asp32,

impedes NADP+ with a 2¢-phosphate group [39] The

corresponding residue in 3a-HSD from P sp B-0831 is

Arg33, which is conserved in the NADP+ preferring

enzymes, such as mouse liver 11b-HSD and mouse lung

CR [16,41] The crystal structure of mouse lung CR

complexed with NADPH displays a pair of basic residues,

Lys17 and Arg39, which correspond, respectively, to Ser11

and Arg33 in P sp B-0831-derived 3a-HSD, causing

electrostatic interaction with the 2¢-phosphate group of

NADP+ [41] These results indicate that the Asp32 and

Arg33 residues are, respectively, critical for NAD+ and

NADP+preferences, resulting in the unique dual nucleotide

cofactor specificity of B-0831 3a-HSD, with the binding

affinity relatively lower than that of the NAD+- or

NADP+-preferring enzyme [7,26,42]

The ordered bi-bi reaction mechanism can be explained

by structural analyses: the substrate-binding loop of

3a-HSD is ordered when nucleotide cofactor binds to the

site next to the substrate-binding site The conformational

change of C testosteroni-derived 3a-HSD/CR induced by

nucleotide binding is more subtle than that of rat liver

3a-HSD [25,39] The conformational change observed in

rat liver 3a-HSD is in good correlation with the slow

formation of a tightly bound complex via a two-step

reaction [26] The NADH binding to P sp B-0831-derived

3a-HSD, which is a two-step reaction, may induce a

conformational change similar to that of rat liver 3a-HSD

In contrast, the binding of NAD+or NADP+, which is a

one-step reaction, may induce little conformational change

similar to that of C testosteroni-derived 3a-HSD/CR The

relatively lower binding affinity of these nucleotides

supports the notion that only a loose complex is formed

in the one-step reaction It should also be noted that the kcat

value for cholic acid as a substrate with NADP+is only 6%

of that for the same substrate with NAD+, although both

nucleotides bind to B-0831 3a-HSD with similar affinity

Additionally, no catalytic activity was detectable with

NAAD+ In short, the different types of nucleotides can

modulate the dynamic conformation and the catalytic

function of the enzyme

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