enzyme-catalysed biotransformations through photochemical regeneration of nicotinamide cofactors

f"3

Enzyme-catalysed biotransformations through photochemical regeneration of nicotinamide cofactors ITAMAR WILLNER and DANIEL MANDLER

Department of Organic Chemistry and the Fritz Haber Center for Molecular Dynamics, The Hebrew University o f Jerusalem, Jerusalem, Israel

Photosensitized regeneration of NAD(P)H cofactors by photochemical means is reviewed. Reductive regeneration of NAD(P)H cofactors proceeds through coupling of photogenerated N,N'dimethyl-4,4'- bipyridinium radical cation, which acts as electron carrier, to the enzymes lipoamide dehydrogenase, LipDH and ferredoxin reductase, FDR, respectively. Regeneration of NAD(P)H is also accomplished by substitution of the enzymes and electron carrier by synthetic rhodium complexes acting as H-donors for the regeneration of NAD(P)H. Various photosensitizers such as Ru(II)-tris-bipyridine, Ru(bpy) 2+, or Zn-meso-tetramethyl-pyridinium porphyrin, Zn-TMPyP 4+, are applied in the systems. The photoinduced processes are initiated through oxidative or reductive electron-transfer quenching pathways. The photoregenerated NAD(P)H cofactors are coupled to subsequent biotransformations in such reduction of ketones and keto-acid, synthesis of amino-acids, and C02-fixation processes. Oxidative regeneration of NAD(P) + cofactors is accomplished by photochemical means. In these systems, Sn(I1)-meso-tetramethylpyridinium porphyrin, Sn-TMPyP 4+, Ru(bpy)~ ÷ or acridine dyes are used as photosensitizers. Oxidation of NAD(P)H proceeds either by reductive quenching of the excited photosensitizer by NAD(P)H or dark oxidation of NAD(P)H by the oxidized photoproduct formed in the photosensitized electron-transfer process. The systems are applied in the dehydrogenation of alcohols, hydroxy acids, and amino acids.

Keywords: Cofactor regeneration; photochemical regeneration of NAD(P)H cofactors; photoinduced biotransformations; photosensitized NAD(P)H regeneration; CO2-fixation; amino acids photosynthesis; photochemical dehydrogenation; photoinduced H2-evolution; biocatalysis; photobiocatalysis; nitrate reduction

Numerous enzymatic reactions require the participation of cofactors in the biocatalytic processes, j-3 These relatively low-molecular-weight (MW < 1500) cocatalysts can be subdivided into two classes: (1) Cofactors that are recycled within the biocatalytic process, and (2) cofactors that require a separate biocatalytic regeneration system that provides the preformed cofactor for the enzymatic transformation. Cofactors recycled within the biocatalytic process include flavin, 4 pyridoxal phosphate, 5 lipoic acid, 6 biotin, 7 and metal porphyrins. 8 For example, oxygen- ases or hydroxylases that contain flavin-type cofactors, transaminases that include pyridoxal phosphate

Address reprint requests to Dr. I. Willner, Department of Organic Chemistry and the Fritz Haber Center for Molecular Dynamics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received 25 July 1988; revised l0 November 1988

as cofactor, carboxylases and decarboxylases that are biotin or lipoic acid dependent, or monoxygenases, peroxidase, and mutase that involve metal porphyrin as cofactors represent catalysts that recycle the cofactor within the process. Oxidation of glucose 9'1° (equation 1) or transamination of pyruvic acid u'~2 (equation 2) demonstrate cofactor-dependent biocatalytic transformations that recycle the cofactor within the process itself.

The main cofactors that require their separate regeneration for sequential biocatalysed processes include 1'13-15 adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), and coenzyme A (CoA). It is estimated that ca. 70% of the enzymatic transformations in nature require the active regeneration of cofactors. Two biocatalytic routes 16A7 for the regeneration of ATP and the subsequent phos-

© 1989 Butterworth Publishers Enzyme Microb. Technol., 1989, vol. 11, August 467

Review

OH

H O ~ OH HO

Glucose oxidase

O:

OH

H O ~ - / ~ H O % O

+ H202 ( l )

+NH3,. H 0

+ C H 3 - - C C 0 2 ~ -OzC" COl

O H 3 N + ~

- O 2 C / ~ ' ~ C O 2 + CH3" "CO2- (2)

phorylation of glucose or glycerol are exemplified in Figure 1, where acetyl phosphate or phosphoenol pyruvate in the presence of acetate kinase or pyruvate kinase, respectively, provides the ATP regeneration cycles.

Oxido-reductase enzymes participate in numerous biotransformations ~8 and involve the participation of NAD(P)+-NAD(P)H cofactors. NAD + and NADP ÷ undergo m'2° a two-electron reduction to form the di- hydronicotinamide cofactors, NAD(P)H:

NAD(P) + + 2e- + H + ~ NAD(P)H;

E ° = -0.32 V vs. NHE (3)

This reduction process is accompanied by a substantial kinetic barrier and the enzymes catalyse these transformations. Thus, sequential biotransformations that are NAD(P)+/NAD(P)H dependent require two coupled enzymes (Figure 2). The regeneration cycles are either reductively generating NAD(P)H (Figure 2a) or oxidatively producing NAD(P) ÷ (Figure 2b) for the subsequent reaction with the substrate.

Several approaches for the regeneration of NAD(P)+/NAD(P)H cofactors, including enzymatic, chemical, and electrochemical methods, have been de-

ADP ATP ( X D P - ~ X T P ; X= U , G , C )

0 0 0 OP DP(O-) z D DP(O-)~ = CH3COP J~,.,~O-

0

ADP~ATP D = CHsCO-"#~'>F "0- 0 ~ < ~ Enzyme = Acetate Pyruvote

Product Reactant Kinase K i nose

Figure 1 Regeneration cycles for ATP cofactor

o....,

Enzyme 1 Enzyme 2 Enzyme 1 Enzyme 2

a b

Figure 2 Schemes for NAD(P)+/NAD(P)H regeneration: (a) biocatalysed transformation through NAD(P)H regeneration; (b) biocatalysed transformation through NAD(P) + regeneration

veloped in recent years and comprehensively reviewed in several reports. 2~-23 Enzymatic regeneration cycles for NAD(P)H cofactors were extensively developed by Whitesides and his collaborators. Reduc- ing agents such as formate 24-26 (in the presence of formate dehydrogenase), glucose 27 (with glucose dehydrogenase), glucose-6-phosphate 28'29 (in the presence of glucose-6-phosphate dehydrogenase), metha- nol 3° (and the enzyme alcohol dehydrogenase) or hydrogen 31-34 (with the enzyme hydrogenase) were effectively applied for the regeneration of NAD(P)H (Figure 3). Whole cells such as chloroplasts or algae that contain NAD(P)H regeneration systems and the coupled enzyme for the desired transformation have also been applied. 35-37 Such intact cells provide immobilized assemblies that contain the cofactor and the coupled biocatalysts. Oxidative pathways (Figure 2b) that use enzyme-aided regeneration of NAD(P) + were also developed. 38-43 For example (Figure 4), a-ketoglutaric acid and pyruvic acid have been applied 38-4~ in the regeneration of NAD(P) + using glutamate dehydrogenase and L-lactate dehydrogenase, respectively, while acetaldehyde was used 42'43 as oxidant of NADH in the presence of alcohol dehydrogenase. Another approach for the oxidative regeneration of NAD + from NADH involves the use of an additional cofactor. Jones and Taylor 44-46 have applied flavin mononucleo- tide (FMN) for the regeneration of NAD + from NADH. In this system the reduced flavin cofactor is reoxidized to FMN by molecular oxygen and the resulting hydrogen peroxide is enzymatically decomposed with catalase.

Electrochemical regeneration of NAD(P)+/ NAD(P)H cofactors is an attractive alternative. Direct oxidation of NADH to NAD + is accomplished 47-49 on Pt electrodes with 90-99.3% recycling efficiency. Im- proved effectiveness was obtained 5°'5t by derivati- zation of the electrode with catechol, or, alternatively, immobilization of the cofactor to the electrode mate- rial. Nevertheless, direct electroreduction of NAD(P) + is usually accompanied by the destructive dimerization of NAD(P); the one-electron reduction product of NAD(P) + to the biologically inactive dimer [NAD(P)]2. 52-55 An alternative route that uses electrogenerated electron transfer mediators for the regeneration of NAD(P)H seems superior, as it suppresses the competitive dimerization process. N,N'-Dimethyl-4,4'-bipyridinium radical (MV +-) acts as an effective electron transfer mediator 56-6° to

468 Enzyme Microb. Technol., 1989, vol. 11, August

HCO0- ~ , f NAD + X Formate Dehydrogenase

CO 2 wr --~ NADH

JOR + NAD(P)

"OH ""OH " ~ / y G6P Oehydrogenase (R= PO{-)

Glucose Oehydrogenase ( R = H )

.o .C"o N o(P,. HO'~ 0

H O ~ COO- OH

NAD (P)+ NAD (P) H

EtOH~CH~COO- Alcohol Oehydrogenose • ! Aldehyde Dehydrogenose

CH~ ClIO

NAD + NADH

M e ~ o z Alcohol Oehydrogenose p / "~ Formate Dehydrooenose

HzCO HCOO- Aldehyde Dehydrogenase

Hz~ ~-2MV2-~, ~ NAD(P)H rm.~na~Y y L/poem/de Dehydrogenose (NAD(H))

Hydro . . . . . . . ~ /~Ferredex,~ Reducio,, (NADP(H))

~.2MV t j ~ NAD (p) +

H2 ~ Fo(ox)'~//'vNADPH

"~Fo(red) / ~ NADP+

Figure 3 Enzyme-catalysed regeneration processes for NAD(P)H cofactors

NADP ÷ and NAD ÷ in the presence of ferredoxin- NADP-reductase (FDR) and lipoamide dehydrogenase (LipDH), respectively (equation 4). The electrochemical regeneration of NAD(P) ÷ has also been em- ployed.

NAD(P) + + 2MV +" + H + FDRor. LioDH

NAD(P)H + 2MV 2+ (4) Electrocatalysed reduction of NAD + to NADH has also been accomplished 6j using an electrogenerated Rh(I)-tris-bipyridine complex. Reduction of NAD ÷ in this system presumably proceeds through a hydrido- rhodium intermediate. The electrochemical regeneration of NADH through the application of Rh(bpy)] + as a redox catalyst is severely limited due to the competitive dimerization of NAD- to (NAD)2. Recently 62 the modified redox catalyst Rh(III)-tris-(2,2'-bipyridyl-5-

Enzyme-catalysed biotransformations: L Willner and D. Mandler

0 -OOC "/~'/a"COO- +NH~, ~ NAD (P) H

+ | Glutamate Oehydrogenaae NH3 J~ -OOC'/"~CO0 -'4/ ~NAD (P)+

0 .,,X,,CO0_ ~ NADH

OH ~-Lactate Dehydrogenase

/~.,,CO0 -,w ~ NAD +

CH3CHO ~ NADH

A/coho/ Oehydrogenase

EtOH ~ "~'NAD +

Figure 4 Enzyme-catalysed regeneration processes NAD(P) ÷ cofactors

for

sulfonic acid) [Rh(bpy-SO3H)] +] has been applied in the electrocatalysed reduction of NADH with substantial success.

Chemical regeneration of NADH has been accomplished with dithionite 63-66 ($20~-) as a reducing agent. Yet, the turnover numbers of the cofactor are low (TN = 105) and the medium severely affects the stability of the added enzymes.

In the present report the regeneration of NAD(P)+/ NAD(P)H cofactors through photochemical means is reviewed. Specific emphasis is directed towards the regeneration of these cofactors by visible light.

Light-active materials that absorb visible light and induce electron transfer processes were extensively studied in the past decade from theoretical 67'6s and practical points of view. 67-72 Organometallic complexes 73'74 such as Ru(II)-tris-bipyridine (Ru(bpy)] + (1), or metalloporphyrins, 75 i.e. Zn-meso-tetramethylpyridinium porphyrin (Zn-TMPyP 4+) (2), and organic dye compounds, i.e. acridine or flavin dyes, 76 have been widely applied as photosensitizers that induce electron transfer reactions. Another type of highly active compounds that induce photosensitized electron transfer processes are semiconductor materials in the form of powders or colloids. In these materials, excitation of valence band (V.B.) electrons to the semiconductor conduction band (C.B.) by light of wavelength exceeding the band-gap energy separation, generates an electron-hole pair (e-, h+), in the respective bands.

Semiconductor materials, such as TiO2, CdS, or FezOs have been widely applied in photosensitized electron transfer transformations. 77-79

Recent advances in the application of photosensitized NAD(P)H regeneration systems in biotransformations are provided in the present review and reveal

Enzyme Microb. Technol., 1989, vol. 11, August 469

Review

broad potential applicability of solar light energy and biocatalysts in bioorganic synthesis.

Photosensitized NAD(P)H regeneration systems A photosensitized NADPH regeneration system is outlined in Figure 5. 8°'s] It involves Ru(II)-tris-bipyridine (1) (Ru(bpy)~ +) as photosensitizer, N,N'-dimethyl-4,4'-bipyridinium, methyl viologen (MV 2+) as primary electron acceptor, ethylene diamine tetraacetic acid triammonium [(NH+)3EDTA] as sacrificial electron donor, the cofactor NADP + and ferredoxin- NADP+-reductase (FDR). Illumination of this system with visible light, h > 400 nm, results in the formation of NADPH through the sequential mechanism displayed in Figure 5. The primary process involves the electron transfer quenching of the excited photosensitizer by MV 2+. The reduced photoproduct, MV +., mediates the reduction of NADP + to NADPH in the presence of FDR, while the oxidized photosensitizer oxidizes the electron donor and as a result the light- active compound is recycled. The quantum yield for

NADPH formation is ¢h = 1.9% and equals that of MV +. production. Thus, the rate-limiting step in this photosensitized NADPH regeneration system is the photochemical step, i.e. production of MV +.. The net photosensitized regeneration of NADPH corresponds to the reduction of NADP + by (NH4)3EDTA (equation 5). The thermodynamic balance of this process reveals that it is an endoergic process by ca. AG = 28 Kcal • mole ~.

R-N(CH2CO~)2 + NADP+-~ R-NHCH2CO2 + CH20 + HCO3 + NADPH + H + (5)

A photosensitized NADH regeneration system is displayed in Figure 5. sl It involves Zn(II)-meso-(N- tetramethylpyridinium)porphyrin (Zn-TMPyP 4+) as photosensitizer, methyl viologen as primary electron acceptor, mercaptoethanol as electron donor, NAD + and the enzyme iipoamide dehydrogenase (LipDH). Illumination of this system results in the production of NADH. The sequential process that leads to the formation of NADH is summarized in Figure 5 and detailed in equations (6-8). The primary step involves the electron transfer quenching of the excited photo-

Oxidation Products

(NH4) 3 EDTA or HO(CH2)2SH

hv / #o# °

*" f N A D H S MV 2+

~ LipDH

NAD +

ADPH

S + MV +" NADP+

R

Zn-TNpyp4+ = R ~ R 2+ Ru (bpy) 3

(Z) (1) R

2+

R -- -CH3 LipDH = lipoamide dehydrogenase

HV 2+ = M e . - - - ~ N ~ N ~ _ M e FDR = ferredoxin-NADP+-reductase

Figure 5 Photosensitized regeneration scheme for NAD(P)H cofactors. The photosensitizer, S, for NADPH regeneration is Ru(ll)-tris- bipyridine [Ru(bpy) 2÷] (1) and for NADH regeneration Zn-meso-tetramethylpyridinium porphyrin (Zn-TMPyP 4÷) (2)



sensitizer, Zn-TMPyP 4+ (equation 6). The resulting oxidized photosensitizer oxidizes mercaptoethanol and thereby the light-active compound is regenerated (equation 7). The reduced photoproduct MV ÷- mediates the reduction of NAD ÷ in the presence of LipDH (equation 8). The net photosensitized transformation corresponds to the reduction

Zn-TMPyP 4+ + MV 2+-~

Zn-TMPyP 5+- + MV +. (6)

Zn-TMPyP 5+. + HSCHzCH2OH ~ Zn-TMPyP 4+

+ 1/2(HOCH2-CH2S-SCH2CH2OH) (7)

2MV +. + NAD + + H+ LipDH.

2MV 2+ + NADH (8)

NAD + + HSCHzCH2OH -~

NADH + I/2(HOCHzCHzS-SCHzCH2OH) (9)

of NAD + by mercaptoethanol (equation 9). The ener- getic balance of this transformation reveals that the process is endoergic (uphill) by ca. AG = 14.5 Kcal • mol -~ and exemplifies an energy storage transformation where light energy is converted and stored in the reduced cofactor, NADH. In contrast to the closely related NADPH regeneration cycle, where the efficiency of NADPH production is determined by the photochemical process, the rate-limiting step in NADH formation involves the electron transfer from the electron carrier to NAD + (equation 8). A limitation of this NADH regeneration cycle involves the poor stability of the photosensitizer, Zn-TMPyP 4+, which undergoes photodegradation in the process.

Semiconductor particles, in the form of powders or colloids, were applied 82'83 as the light-harnessing components which substitute the photosensitizers in photochemical assemblies that regenerate NAD(P)H cofactors. TiO2 and CdS were used as photocatalysts for NAD(P)H cofactor regeneration in systems that include methyl viologen as primary electron acceptor, mercaptoethanol (for TiO2) or formate (for CdS) as electron donors, and the respective enzymes, FDR or LipDH (Figure 6). In these systems photoexcitation of the semiconductor results in an electron-hole pair. Electron ejection of the conduction-band to the electron acceptor generates MV +., which mediates the regeneration of NAD(P)H, while the valence-band hole is scavenged through oxidation of the electron donor. The quantum yields for NADH formation correspond to ~b(TiO2) = 3 × I0 -3 and ~b(CdS) = I x

10 -2 and those of NADPH regeneration are 4)(TIO2) = 1.1 × 10 -3 and qb(CdS)= 1.9 × 10 -3.

A nonenzymatic photosensitized regeneration system for NADH has been reported, 84 using photogenerated Rh(I)-bis-bipyridine as electron transfer reagent. The system is composed of Ru(II)-tris- bipyridine as photosensitizer, Rh(III)-tris-bipyridine (Rh(bpy) 3+) as electron acceptor, and triethanolamine as sacrificial electron donor (Figure 7). Illumination of this system generates, through oxidative electron

COO- l C =0 * NH4 +

MV 2. I H

HCO~ .. " ~ ~ V c, oo-

po- ?o ",.co~ ,5-0. c,.~

CH 2 CO0" CO0"

0 MvZ. /wkH3C'C- CO0-

J \ Y

~H°c"~c"~sH"L el ,~. / Coo- coo- / ~ HzO*H-C-NH 3 @: O'NH;

2H+*KHOCHzCHzS)z CH3 CH 3

Figure 6 Photosensitized regeneration of NADH cofactor using semiconductor particles and colloids, and sequential NADH- dependent biotransformations: (a) lipoamide dehydrogenase (LipDH); (b) alanine dehydrogenase (AlaDH); (c) malate dehydrogenase (MalDH); (d) lactate dehydrogenase (LacDH)

transfer, Rh(bpy) 2+ , which undergoes rapid disproportionation followed by loss of a bipyridine ligand to form Rh(bpy)~-. The latter photoproduct mediates the reduction of NAD + to NADH, probably through an hydrido-Rh(I)-bis-bipyridine intermediate. This system has recently been improved 85 by using-rhodium(III)-tris-(2,2'-bipyridyl-5-sulfonic acid) as the electron relay and hydride transfer catalyst. The origi- nal redox catalyst, Rh(bpy)~ +, exhibits a relatively low reduction potential (-0.92 V vs. SCE) that results in the competitive dimerization of reduced NAD ÷ to the inactive product, (NAD)2, and consequently only limited turnover numbers (ca. 3) for NADH regeneration were observed. This disadvantage was resolved by application of Rh(bpy-SO) 3- as redox catalyst. The reduction potential of the derivatized electron relay is positively shifted (E ° = -0.73 vs. Ag/AgCI) and thus, the destructive reduction of NAD ÷ to NAD- and

Y T~oe,-k A

~'~ [RuIboy)~] ~®I \ Z[R, Cbpy)f" TEOA

bpy

J

boy ~ H °

Figure 7 Photosensitized NADH regeneration through a rhodium homogeneous catalyst


Review

subsequent dimerization to (NAD)2 is diminished. The system where NADH is regenerated with Rh(bpy- S03)~- as catalyst has been coupled to the subsequent reduction of cyclohexanone to cyclohexanol using horse liver alcohol dehydrogenase (HLADH). Re- markably high turnover numbers for NADH regeneration (ca. 40) and the redox catalyst Rh(bpy-SO3)~- (ca. 80) were observed.

Photosensitized NAD(P) + regeneration systems

Photoinduced oxidative regeneration of NAD(P) + from NAD(P)H can be initiated through two alternative pathways (Figure 8). NAD(P)H might participate in the photochemical reaction and act as a quencher for the excited sensitizer. By reductive electron transfer quenching of the excited light-active compound, NAD(P) + and the reduced photosensitizer are formed (Figure 8a). The reduced photosensitizer can then mediate the dark reduction of an electron acceptor, a process which regenerates the light-active compound. The second photochemical route for the regeneration of NAD(P) + involves the oxidative electron transfer quenching of the excited photosensitizer by an electron acceptor (Figure 8b). The resulting oxidized photosensitizer mediates the dark oxidation of NAD(P)H and thereby the light-active compound is regenerated.

The photosensitizer Sn(IV)-meso-N-tetramethylpyridinium) porphyrin, Sn-TMPyP 4+, has been applied 86 as photosensitizer for the regeneration of NAD ÷ from NADH through the reductive quenching route. The triplet excited state of Sn-TMPyW + is quenched via electron transfer to form the metalloporphyrin 7r-radical anion, Sn-TMPyP 3+- and the oxidized photoproduct NAD. (equation 10). The metalloporphyrin radical anion Sn-TMPyP 3÷- undergoes either disproportionation (equation 1 1) or dark oxidation by NAD. (equation 12) to form the metal chlorin, the protonated metalloporphyrin dianion.

*Sn-TMPyP 4+ + NADH

Sn-TMPyP 3+. + NAD- + H + (10)

The oxidized photoproduct NAD- is also capable of being reduced by Sn-TMPyP 4+ (equation 13). Conse- quently, the two-electron oxidation of NADH is pho-

hv hv

NAD(P) + S- A NAO(P)H S +

(a) (b) Figure 8 Schemes for the photosensit ized regeneration of NAD(P) + cofactors: (a) through a reductive quenching mechanism; (b) through an oxidat ive quenching process. S is the photosensitizer and A- the electron acceptor

H2

SnTMPyPH3*~NAD*) /C-zHsOH SntMPyph~/ /. YADH "NADH ,v ACH3CHO +H +

R

Sn-TMPyP 4+= R ~ R

R R= --~1~- CH 3

Figure 9 Photosensitized H2 evolut ion through the NAD + re- generat ion

tochemically derived by Sn-TMPyP 4+ (equation 14). The reduced chlorin derivative Sn-TMPyPH 3÷ mediates the reduction of proton to hydrogen in the presence of colloidal Pt (equation 15), a process that recycles the photosensitizer.

2Sn-TMPyP 3+- + H + ~__

Sn-TMPyP 4+ + Sn-TMPyPH 3+ (11)

Sn-TMPyP 3+- + NAD- + H+--~

Sn-TMPyPH 3+ + NAD + (12)

Sn-TMPyP 4+ + NAD.

Sn-TMPyP 3+. + NAD + (13)

Sn-TMPyW + + NADH-%

Sn-TMPyPH 3+ + NAD + (14)

This allows the cyclic oxidation of NADH, formed by oxidation of ethanol in the presence of yeast alcohol dehydrogenase (YADH), through concomitant H2- evolution as outlined in Figure 9. The process is driven by visible light, X = 550 nm, and proceeds with a quantum efficiency of d, = 0.43.

Sn-TMPyPH 3+ + H+-+

Sn-TMPyP 4+ + H2 (15)

Julliard and Le Petit 87 have applied organic dyes such as methylene blue (MB ÷) or N-methyl phenazonium methyl sulfate (MPMS +) as photosensitizers for the light-induced regeneration of NAD ÷ and NADP ÷ through the reductive quenching mechanism. Other dyes, 88 such as Rose Bengal, eosin, 2,6-dichlorophenol indophenol, and thionin, were also reported to be active photosensitizers in this process. The excited states of MB + and MPMS + are powerful oxidants (E°(*MB+/MB) = 1.61 V; E°(*MPMS÷/MPMS) =



2.53 V vs. SCE) and therefore capable of oxidizing NAD(P)H. The photosensitized regeneration of NAD(P) ÷ using MB ÷ and MPMS ÷ as light-active compounds is schematically presented in Figure 10. In these cycles the primary step involves the reductive quenching of *MB + or *MPMS + by NAD(P)H (equation 16). The resulting MB. or MPMS- are rapidly protonated (equation 17) and undergo thermal reduction by NAD(P). to the fully reduced leuco forms of methylene blue and N-methyl phenazonium (MBH2, MPMSH2) (equation 18), respectively. The leuco products are reoxidized by molecular oxygen to MB ÷ and MPMS ÷.

*MB ÷ (or *MPMS +) + NAD(P)H --*

MB. (or MPMS.) + NAD(P)- + H ÷ (16)

MB- (or MPMS-) + H+ --->

MBH ÷. (or MPMSH +.) (17)

MBH- (or MPMSH.) + NAD(P)- + H + --~

MBH2 (or MPMSH2) + NAD(P) ÷ (18)

The quantum yields for NAD + regeneration with MB + and MPMS ÷ correspond to ~b = 0.3 and ~b = 0.28, respectively. A closely related system 89 applies acridine orange immobilized to sepharose (3) as light-harnessing compound for the regeneration of NAD(P) +. The mechanism of NAD(P) ÷ regeneration is similar to that described for MB ÷ or MPMS +. In this system the enzyme catalase is added to decompose hydrogen peroxide formed in the cycle.

Immobilized acriflavin =

CH3

o'. (3)

Oxidative regeneration of NAD + has been accomplished 9° by using Ru(II)-tris-bipyridine (Ru(bpy)~ +) as photosensitizer. In the presence of methyl viologen (MVE+), oxidative quenching of the excited photosensitizer leads to the formation of MV +. and the oxidized photoproduct, Ru(bpy)~ +. The latter photoproduct is reduced by NADH, a process that recycles the light- active compound and regenerates NAD +. The resulting MV +- mediates HE-eVolution in the presence of colloidal Pt. Hence, the net process corresponds to the light-induced regeneration of NAD + from NADH through HE evolution (equation 19) and proceeds with a quantum efficiency of ~b = 0.3%.

NADH + HEO -~ NAD + + HE + OH- (19)

Photosensitized biotransformations through NAD(P)H regeneration The photosensitized regeneration cycles of NAD(P)H cofactors allow the introduction of specific enzymes

NAD + I MBH 2 Subsh'ote AH 2 ~ ~ or Jhl/ or ~.----202

Oxidized ~ "~.NADH / X. MB+ . / \ +

NADPH MPMS ÷ " ' 2 ~ ' ~Zv~÷~Z

L MB + or MPMS +

Methylene blue X=S R = N (CH 3 )2

N - methylphenazo~nlum

methyl sulfato X=N

R=H

Figure 10 Photosensitized regeneration of NAD(P) + through reductive quenching and application of oxygen as sacrificial electron acceptor

dependent on these cofactors and the performance of a variety of "one pot" biotransformations. The NADPH regeneration cycle has been coupled 8°'8j to the reduction of 2-butanone in the presence of NADPH-dependent alcohol dehydrogenase (AIDH) (from T. Brocii) and to the reductive amination of a-ketoglutaric acid with glutamate dehydrogenase (GIuDH) to form glutamic acid (Figure 11). The rate of 2-butanol and glutamic acid production as a function of illumination time is displayed in Figure 12 and the production efficiency of 2-butanol is examined under long-term illumination. It is evident that the productivity of such systems is maintained as long as the sacrificial electron donor that recycles the photosensitizer is present in

MVZ~ NADPH b" CH3 CHz~C~ H~

'X~/4~ ~CH3CHz!_CH 3

7 ; 7 MV ~ N A O P ~

coo- .

CO0- C=O÷NHa H2 0,,- H- ,C-IN H 3 ,CHz

COO- CHz C00-

Figure 11 Biotransformations coupled to the photosensitized NADPH regeneration cycle: (a) ferredoxin-NADP+-reductase (FDR); (b) alcohol dehydrogenase (AIDH); (c) glutamic acid dehydrogenase (GluDH)


Review

~ 3

g2

A

• =2-~=,a~ol

i I0 2JO 3~0 4C

Illumination time (hours)

/ 3 ~

% ! /"

J i" i = ,ac, ,c ac ,~

5 I0 [lluminatioq time (hours)

Figure 12 Rates of product formation in various photosensitized biotransformations upon illumination of (A) NADPH and (B) NADH regeneration systems. In (A) the sacrificial electron donor (NH4)aEDTA is added at (a), (b), and (c)

the system. Its readdition to the system, upon con- sumption, regenerates the activity of 2-butanol formation.

Glutamic acid provides a natural synthon for the synthesis of other amino acids. The photosensitized NADPH regeneration cycle has been applied 9j for the sequestered photoinduced synthesis of aspartic acid as well as alanine (Figure 13). In this cycle, Ru(bpy)23 + is used as photosensitizer and the electron transfer- mediated process yields NADPH and the primary product, glutamic acid. This synthone mediates the specific transamination processes and yields aspartic acid (equation 20) or alanine (equation 21) in the presence of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT), respectively.

N H f I

HO2C-CHCHzCH2COzH +

0 II

HO2C-CCH2CO2H . GOT

O NH~ II I

HO2C-CCH2CH2COzH + HOzC-CHCHzCO2H (20)

N H f O I II Gvr

HOzC-CH-CH2CH2COzH + CH3CCOzH- "

O NH~ II I

HO2CCCH2CH2CO2H + CH3-CH-CO2H (21)

Table 1 summarizes the turnover numbers of the artificial components and biocatalysts in these transformations. These data reveal that the components are effectively recycled and the systems exhibit high stabilities.

~H, ,NH~ h -O~CCHzCHCO ~

2(OCHzCHzS) z /~.-~u(bgy)3 MV NAOPH -0 C H CC0- / ~H3 "~1 ~ I J - X ~ ~ / - = ~, ,~>/pc.,:.co;

.~ t 01 4 . . . . ~ o J , .., ,%. ,0c.,:.=s, ~-e, b0,,'J~---~v* J ~ ..o.,_#L,~ . . . . ;oo_j, c,,~c~;

-0zC2HzCSO ~

Figure 13 Multienzyme-catalysed biotransformations using the photosensitized NADPH system: (a) ferredoxin-NADP +- reductase (FDR); (b) glutamic acid dehydrogenase (GluDH); (c) glutamic oxaloacetic transaminase (GOT); (d) glutamic pyruvic transaminase (GPT)

The photosensitized NADH regeneration cycle using Zn-TMPyP 4+ as photosensitizer (Figure 5) was also coupled 81 to subsequent biotransformations (Fig- ure 14). Reduction of pyruvic acid to lactic acid using lactate dehydrogenase (LacDH), reductive amination of pyruvic acid to alanine with alanine dehydrogenase (AiaDH), and reduction of acetoacetic acid to /3-hydroxybutyric acid in the presence of hydroxybutyrate dehydrogenase (HButDH). The rates of product formation are depicted in Figure 12b. Table 2 summarizes the turnover numbers of the various components, conversion yields, and optical purities involved in the different reactions using the photosensitized NADPH and NADH regeneration cycles. Since the turnover numbers were reported under conditions where the systems still exhibit activity, these should be con- sidered as lower limits implying substantial stability of biocatalysts and synthetic ingredients in these hybrid systems. The data provided in Table 2 reveal also that the optical purity of products is high.

The net reactions outlined in Figure 14 correspond to the light-induced reduction of pyruvic acid and acetoacetic acid to lactic and/3-hydroxybutyric acids by mercaptoethanol (equations 22 and 23), and to the reductive amination of pyruvic acid to alanine by mercaptoethanol (equation 24). The thermodynamic balances of these reactions are also given and reveal that the production of lactic acid and /3-hydroxybutyric acid are endoergic processes. The endoergic

hu 0

k . f HsS-~-COO-

e 0

COO- CO0- H20*H-C- NH ~ C=O÷NH;

CH 3 CH 3

Figure 14 Biotransformations coupled to the photosensitized NADH regeneration cycle: (a) lipoamide dehydrogenase (LipDH); (b) lactate dehydrogenase (LacDH); (c) /3-hydroxybutyrate dehydrogenase (/3-HButDH); (d) alanine dehydrogenase (AlaDH)

474 E n z y m e M i c r o b . T e c h n o l . , 1989, vo l . 11, A u g u s t


Table 1 Turnover numbers of the components in the different enzyme-catalysed reactions

Ru(bpy)~ + MV 2÷ NADP ÷ FDR" GluDH b GOT c or GPT d

Glutamic acid 1220 31 16 2.6 x 104 7.4 x 108 Aspartic acid 1480 37 19 3.1 x 104 9 x 106 GOT 1.5 x 104 Alanine 1960 39 20 3.3 x 104 9.4 × 108 GPT 1.9 x 104

" Formula weight (F. W.) 40,000 (M. Shin, Methods EnzymoL, 1971, 23, 441). b F. W. 2,200,000 (H. Surd and W. Burchard, Eur. J. Biochem., 1968, 6, 202). c F.W. 110,000 (W. T. Jenkins, D. A. Yphantis, and I. W. Siger, J. Biol. Chem., 1959, 234, 51 ). d F. W. 115,000 (M. Saier, Jr., and W. T. Jenkins, J. BioL Chem., 1967, 242, 91)

balance of the photosensitized biotransformations using the NAD(P)H regeneration cycles exemplifies the potential advantages of photochemical regeneration means over chemical regeneration.

O II

CH3CCO2H + 2HOCH2CH2SH - ~

OH I

CH3CHCO2H + (HOCH2CH2S)2

AG = 7 Kcal • mole -j (22)

O II

CH3-CCH2CO2H + HOCH2CH2SH

OH I

CHaCHCH2CO2H + (HOCH2CH2S)2

AG = 15 Kcal • mole -j (23)

O II

CH3CCO2H + NH~ + 2HOCH2CH2SH

CH3CHCO2H + H +, + H20 I

NH2 AG -~ 0 (24)

This aspect is exemplified in Figure 15 for the synthesis of alanine by chemical regeneration of NADH using ethanol as H-donor and alcohol dehydrogenase (AIDH) (Figure 15a) and the light-induced synthesis through photochemical means (Figure 15b). In the chemical regeneration scheme, product accumulation is controlled by the specific thermodynamic balance of the process (equation 25). Since all sequential steps exhibit reversibility, the system reaches equilibrium as a result of the reverse processes, namely, oxidation of alanine through acetaldehyde reduction.

O II

CH3CCO2H + NH~- + CH3CH2OH

NH2 I

CH3CHO + CH3-CHCO2H + H20 (25)

As a result, reactions that exhibit an unfavored thermodynamic balance will allow only limited amounts of product to accumulate. These difficulties might be resolved by increasing the concentration of the reduc- tant, which might be harmful to the enzymes, or by the application of H-donors whose oxidized product can be eliminated from the system, i.e. formate being oxidized to CO2.

The photochemical regeneration of NAD(P)H cofactors provides a general alternative approach to drive endoergic NAD(P)H dependent syntheses. In such biotransformation, light energy is converted into chemical energy that is stored in the reduced relay (MV +.) and reduced cofactor [NAD(P)H]. As a result, quantitative accumulation of product, upon illumination, is envisaged with no interference of the product reoxidation process.

CO2 Fixation through photosensitized N A D ( P ) H regeneration cycles

Carbon dioxide plays a central role in the universe cycle 92-94 (Figure 16). It provides the carbon source for the photosynthetic system resulting in carboxylation products that subsequently undergo complex bio- synthetic transformations. The resulting organic compounds provide the essence for the heterotrophic cycle

o) Chemical Regeneration NH2 I CH3CH2OH I~/~NAD+ ~ ' ~ C H 3 CHCO2 H (3)

C H 3CH O ' ~ r " N A D H ~ CH3COC02 H (1)

AI cDH AIODH + NH +

b) Photochemical Regeneration

hu fCH, C.CO,. (S)

@ N.. V' -~"JX '~NADH~ZI~ CH~COCO z H ( 1 )

LipOH AIoDH ÷ NH~

Figure 15 Chemical and photochemical regeneration of NAD(P)H: (a) chemical regeneration and application in synthesis; (b) photochemical regeneration and application in endoergic synthesis


Review

T a b l e 2 T u r n o v e r n u m b e r s (TN) o f t h e c o m p o n e n t s in t he d i f f e r e n t e n z y m e - c a t a l y s e d r e a c t i o n s

Final Initial % product substrate

Enzymes % Optical concentrat ion concentrat ion Ru(bpy)~ ÷ ZnTMPyP 4. MV 2~ NAD(P) + ~ Conversion puri ty (M) (M)

Butan-2-ol 530 40 40 FDR a AIDH c 27 100 +- 10 4.05 x 10 2 0.15 2.4 x 104 6 x 103

Glutamic acid 225 15 15 FDR GluDH a 13 99 -+ 7 1.3 x 10 2 0.1 9.75 x 103 1.26 × 105

Lactic acid 4,500 13.5 27 LipDH b LDH e 27 94 -+ 7 2.7 x 10 2 0.1 5.67 x 104 4.15 x 104

Alanine 7,900 19 27 LipDH AlaDH f 38 78 -+ 7 3.8 x 10 -2 0.1 1.7 × 104 3.2 x 106

/3-Hydroxybutyric 1,900 2.4 10 LipDH /3-HBuDH g 21 105 -+ 10 9.4 x 10 3 4.52 x 102

acid 5.2 x 103 8.3 × 10

a F. W. = 40,000 (M. Shin, Methods Enzymol, 1971,23, 441 ). b F. W. = 70,000 (F. B. Straub, Biochem. J., 1939, 33, 787). c F. W. ~ 40,00011 a F. W. = 2,200,000 (H. Sund and W. Burchard, Eur. J. Biochem., 1968, 6, 202). OF. W. = 140,000 (R. Jaenicke and S. Knof, Eur. J. Biochem., 1968, 4, 157). fF. W. = 228,000 (A. Yoshida and G. Freese, Biochim. Biophys. Acta, 1964, 92, 33). Q F. W. = 850,000 (H. U. Bergmeyer. Biochem. J., 1967, 102,

423)

where energy is evolved through the respiration chain (decarboxylation).

Carbon dioxide fixation processes can be classified by several approaches. 95 The nature of the resulting reduced carbon bond provides one means of classification. Carboxylation products through C-C bond formation (equation 26) or carboxylation products that yield nitrogen-carbon, namely, carbamate products (equation 27), exemplify this classification approach. For example, carboxylation of urea in the presence of ATP and the enzyme urea carboxylase yields carbamate (equation 28). A second division of CO2 fixation processes is based on the definition of spontaneous carboxylation processes and nonspontaneous cofactor regenerated-dependent transformations.

R - C - H + C02 (or HCO3) --> R-C-CO~ (26)

R - N - H + C O 2 (or HCO3) ~ R - N - C O ; (27)

NH2 Mg 2+, K +, biotin

O~-~C + ATP + HCO~ . " urea, carboxylase

\ N H 2

NH2 /

O~-~C + ADP + pO34 - (28) \

NHCO2

The primary step in photosynthesis, namely, CO2 fixation into ribulose-l,5-diphosphate catalysed by ribulose-l,5-diphosphate carboxylase (RuBP carboxylase), which yields 3-phosphoglycerate 96'97 (equation 29) or the synthesis of oxaloacetate from phosphoenol pyruvate (PEP), catalysed by the respective PEP carboxylase 93"94 (equation 30), are spontaneous CO2

fixation processes. Other CO2 fixation processes require the participation of cofactors such as reduced ferredoxin, ATP, or NAD(P)H. 95

C H ~ - O - P O ~ - I

C = O C O 2 H ) I

H - C - O H + CO2 Mg2+ > 2 H - C - O H I RuBP I

H - C - O H c.,boxylase CH2OPO~-

CH20-PO~- (29)

C O 2 H I

C H 2 C H 2 II I

CO2 + C-OPO]- Mg2+ )' C•O [ PEP, carboxylase ]

C O 2 H C O 2 H

+ p o 3-

(30)

Oxaloacetate is formed through carboxylation of pyruvic acid in the presence of ATP cofactor and pyruvate carboxylase 98 (equation 31) and carboxylation of acetyl-CoA proceeds in the presence of reduced ferredoxin and pyruvate synthase (equation 32).

CH3 ] Mg 2+ or Mn 2+

C=O + ATP + H C O 3 . biotin, pyruvate

[ carboxylase co2

c o i I

CH2 + ADP + po34 - I

C -O I

co2 (31)


O II

Fdred + CO2 + CH3C-CoA pyruvate synthase

O II

Fdox + CH3C-CO2 + CoA


0 II

CH3-C-CO2H + C O 2 + NADPH malic enzyme)

OH I

HO2C-CH-CH2CO2H + NADP ÷ (34) (32)

NAD(P)+/NAD(P)H cofactors play a major role in biological carboxylation-decarboxylation processes. For example, decarboxylation of isocitric acid to ketoglutaric acid, which is a part of the heterotrophous Krebs cycle, proceeds in the presence of isocitrate dehydrogenase (ICDH) and requires the concomitant reduction of NADP ÷ (equation 33). In turn, carboxylation of pyruvic acid, which controls the supply of malic acid to the Krebs cycle, requires the NADPH cofactor and occurs in the presence of the malic enzyme (equation 34). The capability to drive endoergic reactions by light-induced NAD(P)H-cofactor regeneration systems has been used in the development of photoinduced CO2 fixation biotransformations through carboxylation routes.

OH CO2H I I

HO2C-CH-CH-CH2CO2H + NADP + ICDH> Mn 2+

(33)

O II

HO2C-C-CH2CH2CO2H + NADPH + CO2

Photosensitized carboxylation of pyruvic acid or ketoglutaric acid to form malic and isocitric acids has been accomplished 99 in the presence of the malic enzyme and ICDH, respectively, by applying the light-induced NADPH regeneration system (Figure 17). Ru(II)-tris- bipyridine is used as photosensitizer and MV 2+ mediates the regeneration of NADPH at the expense of mercaptoethanol (Figure 5). Photogenerated NADPH mediates the carboxylation processes in the presence of the respective enzymes. The photosynthesized malic acid has been coupled to sequential biotransformations.n°° In the presence of fumarase and aspartase, dehydration of malic acid to fumaric acid followed by amination of the product to aspartic acid occurs (Fig- ure 17). The turnover numbers of the various components are summarized in Table 3 and reveal the stabilities of the assemblies composed of the artificial components and biocatalysts. It was found that the enzyme ICDH exhibits improved stability upon immobilization on a polyacrylamide matrix :°~ in the photosensitized synthesis of isocitric acid. The thermodynamic balance for the photosynthesis of malic acid and isocitric acid through oxidation of thiols is outlined in equations (35) and (36). It is evident that the formation of malic acid is endoergic by AG = 11.9 Kcal • mole -j.

photosynt hesls

I¢,-fi x n tion

mankinds c iv i l izat ion

[ industry)

fossilic o~anic

compounds

_ . . . . . . .

l Krebs cycle organic cycle

Succ - - 12 0"6' / ~-'~1F'6 - P '~'1" T'3"P compounds

• I I 6(ADP,,-Pi| / 6 ~

12(AOP+Pi ] 1'2 ATP

Fauna | Flora 6H70 2 LO2 (,H20 (heterotroph) ~ . J J |outotroph)

photosynthesis

6[I-P]

6ATP

hV

Figure 16 Scheme of the biological photosynthetic and heterotrophic cycles. The following abbreviations are used: ADP, adenosine diphosphate; ATP, adenosine triphosphate; Pi, inorganic phosphate; F-6-P, fructose-6-phosphate; PGA, phosphoglyceric acid; NADP+/NADPH, nicotinamide adenosindinucieotide phosphate couple; R-5-P, ribulose-5-phosphate; R-1,5-DP, ribulose-l,5-diphosphate; T-3-P, triose-3-phosphate; a-Kglu, a-ketogiutarate; Cit, citrate; Fum, fumarate; (H), metabolically bond hydrogen; ISC, isocitrate; Mal, malate; OA, oxaloacetate; Succ, succinate; Succ-CoA, succinate bound to coenzyme A


Review

Table 3 Turnover numbers of the components in the various CO2-fixation reactions

Malic [Ru(bpy)3] 2+ MV 2+ NADP + FDR a FDH b enzyme c ICDH d Fumarase e Aspartase f

Malic acid 1,074 117 62.2 2.3 x 104 7.4 x 10 ~ Aspartic acid 174 25 6.3 1.6 x 103 6.3 × 104 Isocitric acid 272 23 11.4 2.5 x 10 a Formic acid 67 2 2 x 103

5.5 x 104 8,5 x 102 5.2 x 102

a Formula wt. (F. W.) 40,000 (M. Shin, Methods Enzymol., 1971,23, 441 ). b F. W. 300,000 (T. Hopner and A. Trutwein, Z. Naturforsch., Teil B, 1972, 27, 1075). c F. W. 280,000 (R. Y. Hsu and H. A. Lardy, J. Biol. Chem., 1967, 242, 520). d F. W. 58,000 (R. F. Colman. J. BioL Chem., 1968, 243, 2454). eF. W. 48,500 (S. Beckmans and L. Kanarck, Eur. J. Biochem., 1977, 78, 437). fF. W. 48,500 (S. Suzuki, J. Yamaguchi, and M. Tokushige, Biochim. Biophyn. Acta, 1973, 321,369)

O JI hv

CH3CCO2H + CO2 + 2HO-CH2CH2SH--->

OH I

HOzC-CHCHzCO2H + (HO-CH2CH2S)2 (35)

O SH HS

HO2CCCH2CHzCO2H + C02 + --->

HO OH

OH CO 2 S-S

l/ HO2C - C H - CH-CH2COzH +

HO OH

(36)

CO2 and NO~ fixation through enzyme-catalysed photosynthesis

The enzyme formate dehydrogenase (FDH) catalyses the oxidation of formate to carbon dioxide and concomitant reduction of NAD +1°2 (equation 37).

HCO~- + NAD + FDH> CO2 + NADH (37)

Although the process is reversible, the oxidation path-

way is favored (AG o = -4 .6 Kcal • mole-l). The enzyme appears to be nonspecific and various reduced bipyridinium salts j°3 can substitute the natural cofactor, NADH, and mediate the reduction of CO2 to formate. A series of bipyridinium sal ts--N,N'- dimethyl-4,4'-bipyridinium (MV2+), N,N'-dimethyl- 2,2'-bipyridinium (DM2+), N,N'-trimethylene-2,2'- bipyridinium (DT2+), or N,N'-tetramethylene-2,2'- bipyridinium (DQ2+)--have been applied j°° as electron acceptors in photochemical assemblies that include Ru(II)-tris-bipyridine (Ru(bpy)] +) as photosensitizer and cysteine as sacrificial electron donor. Upon illumination, the photogenerated bipyridinium radical cations (V +.) mediate the reduction of carbon dioxide to formate in the presence of the enzyme formate dehydrogenase (FDH) (Figure 18). The highest quantum yield that corresponds to q~ = 1.6% was observed with MV 2+ and DT 2+ acting as electron acceptors. The net photosynthesis of formate through reduction of CO2 by cysteine (equation 38) is an endoergic process, AG = 5.5 Kcal • mole -~.

+NH3 2 ( O 2 C - C H - C H 2 C H 2 S H ) + CO2

+NH3 I (-O2C-CH-CH2CH2S)2 + HCO2H (38)

Very recently photoreduction of nitrate (N03) to nitrite (N02) and of N02 to ammonia has been

O i i

hv / CH3CCOzH ÷ C02

(RS SR) Ru(bpy) 2÷ MV 2÷ NADPH OH e

/ ¢ ° ) . ? / \ 3 . - ~ ~ + / \ cM"-H02 CCHzCH2 CC02H + CO2

RSH" \Ru(bpy)3 ~'K" " ~ M ~ I /ADP~( u H OH ÷/ HCO~ ~.. , '

H *CO z z HOzCCHzC-- CCO 2 H HO2C H

f = HO z CCCHzCOzH

H

Figure 17 Photosensitized biocatalysed CO2 fixation through photoinduced regeneration of NADPH: (a) ferredoxin-NADP+-reductase (FDR); (b) malic enzyme; (c) isocitrate dehydrogenase (ICDH); (d) formate dehydrogenase (FDH); (e) fumarase; (f) aspartase

478 Enzyme Microb. Technol. , 1989, vol. 11, Augus t

1.0

X

=o

0.5

0.4

0.3

0.2

0.1

e-DT 2+ wHhoul FDH electron donor

z~-MV 2+ M e + ~ ' t - Me

&'DTZ+ C ~

E* (Volts)

-0.44

-0.55 + \ / +

(Ell2) 5

V-DQ 2+ ~ -0.65 & +\ /+ /

(CH2)4 / o-DM 2+ ~ -0.72 /

+, ,+ / //A Me Me . /

25 50 75 Tlluminolion lime (min)


IO0

Figure 18 Rates of formate production upon illumination of photosystem in the presence of formate dehydrogenase (ForDH) and various bipyridinium charge relays

accomplished by enzymatic catalysis. J°4 This process might be an important step towards the photoinduced reductive fixation of NO3 to ammonia. The photochemical assembly for NO~ reduction is composed of Ru(bpy)~ + as photosensitizer, methyl viologen (MV 2+) as primary electron acceptor, and (NH4)3EDTA as sacrificial electron donor. Illumination of the system results in the reduced photoproduct MV ÷., which mediates the reduction of NO3 to nitrite in the presence of nitrate reductase, and the reduction of NO~ to ammonia in the presence of nitrite reductase. The reduction process proceeds with a quantum efficiency of ~b = 3%, and high turnover numbers for the synthetic components as well as biocatalyst are observed.

Photosensitized biotransformations through N A D ( P ) ÷ regeneration

The various photosensitized NAD ÷ regeneration cycles have been applied 86 in the oxidation of ethanol in the presence of alcohol dehydrogenase (AIDH). With Sn(IV)-meso-tetra(N-methyl pyridinium) porphyrin, Sn-MPyP 4÷, and Ru(bpy)~ + as photosensitizers, the complementary reduction process corresponds to the reduction of protons and H2 evolution (Figure 9). The net process in these biotransformations corresponds to

the dehydrogenation of ethanol to acetaldehyde (equation 39) through the photosensitized regeneration of NAD ÷. This process is endoergic by 10 Kcal • mole - 1:

C2HsOH ~ CH3CHO + H2

AG°~10 Kcal • mole -1 (39)

The systems that apply methylene blue (MB ÷) N-methyl phenazenium methyl sulfate (MPMS+), or immobilized acridine orange and oxygen as the regeneration reagent of the photosensitizer (Fig. 10) were coupled 87'88 to the A1DH oxidation of ethanol. In these systems ethanol is oxidized to acetaldehyde while oxygen is reduced to hydrogen peroxide (equation 40). Since hydrogen peroxide is further decomposed to oxygen (equation 41), the net reaction involves the oxidation of ethanol by molecular oxygen through the operation of the photosensitized NAD ÷ regeneration cycle (equation 42).

CH3CH2OH + O2--) CH3CHO + H202 (40)

2H202--~ 2H20 + 02 (41)

2CH3CH2OH + O2 ~ 2CH3CHO + 2H20 (42)

Table 4 summarizes the quantum yields for ethanol oxidation as well as the turnover numbers of the cofactor and A1DH in the various systems. The photoinduced NAD ÷ regeneration system using Ru(bpy) 2÷ as photosensitizer has been also applied 9° to the oxidation of lactic acid to pyruvic acid using lactate dehydrogenase (LDH) (equation 43) and to the oxidation of alanine to pyruvic acid in the presence of alanine dehydrogenase (AlaDH) (equation 44).

OH O I II

CH3-CH-CO2H----~ CH3CCOzH + 1/2H2

AG O = 10 Kcal • mole -1 (43)

Table 4 Ethanol oxidation through photosensitized NAD + regeneration

Turnover number

Photosensitizer a Quantum yield (%) NAD ÷ AIDH

Sn(IV)TMPyP 4+ 43 86 1.3 × 10 s Ru(bpy)~ + 0.3 8 1.3 × 104 MB ÷ 3 187 ~104

(1125) b Acridine orange

(immobilized) c 3.1 d

"Precise photochemical configuration is detailed in text b Reported under other experimental conditions c Not specified d Turnover number per 2 h


Review

a- SH : ethanol

b - S H : l a c l i c acid

c- SH : olonine

C e V ~ C 8 V ~ 8V

H Ph Br Br

Phase Ph / \ H H " H h

Figure 19 Application of sepharose-immobilized NAD + photoinduced regeneration system in chemical transformations in organic solvents, i.e. debromination of rneso-dibromostilbene

O II

CH3-CHCO2H + H20 ~ CHs CCO2H + 1/2H2 I

NH2

+ NH3 AG O = 14 Kcal • mole -I (44)

The photosensitized NAD + regeneration system, which uses Ru(bpy)] + as photosensitizer, has been applied 1°5 for the debromination of meso-l,2-dibromostilbene. An oil-water two-phase system, where the NAD+-regeneration system is embedded in sepharose beads, has been used as the reaction medium (Figure 19). The sepharose beads include the photosensitizer (Ru(bpy)~+), N,N'-dioctyi-4,4'-bipyridinium (C8V 2+) as electron acceptor, NAD +, and one of the following substrates with the respective enzyme: ethanol or propanol and AIDH, lactic acid and LDH, or alanine and AIaDH. The sepharose beads are suspended in a toluene phase that includes meso-dibromostilbene. The substrate and respective enzyme mediate the production of the electron donor, NADH, for the photochemical process. Upon illumi-

nation of the system, the reduced photoproduct, CsV +., exhibits hydrophobic properties and is ex- tracted to the organic phase. The oxidized photoproduct, Ru(bpy)~ +, regenerates NAD + and the light- active compound is recycled. In the organic phase, the reduced photoproduct undergoes induced disproportionation through the presence of an oil-water two- phase system. The two-electron reduction product, thus formed, affects the debromination of meso-l,2- dibromostilbene to trans-stilbene. The net photosensitized transformations accomplished in this two- phase system correspond, for example, to debromination of meso-l,2-dibromostilbene and oxidation of an alcohol (equation 45) or oxidation of alanine (equation 46) through the operation of the photoinduced regeneration of NAD+-cofactor.

Br Br I I

CH3CH2OH + Ph- C H - C H - P h

CH3CHO + Ph-CH=CH-Ph + 2Br- (45)

Br Br I I

CH3-CH-CO2H + Ph-CH- CH-Ph I

NH2

O II

CH3CCO2H + Ph-CH=CHPh + 2Br- (46)

The photosensitized NAD + and NADP + regeneration system that uses sepharose-immobilized acriflavin has been applied 89 to a variety of biotransformations, including the oxidation of lactic acid to pyruvic (with LDH), oxidation of ethanol to acetaldehyde (with AIDH), conversion of malic acid to oxaloacetic acid (with MDH), and oxidative deamination of alanine to pyruvic acid (with GIuDH). Table 5 summarizes the kinetic and productivity properties of these biotransformations. The net reactions in these cycles involve the oxidation of the substrates by molecular oxygen, i.e. oxidation of malic acid to oxaloacetic acid (equation 47) or oxidative deamination of glutamic acid to ketoglutaric acid (equation 48).

Table 5 Kinetic and productivity properties for biotransformations involving acriflavin-sepharose photosensitized NAD(P) + regeneration

Substrate Enzyme

NAD(P)H Product formed TN

nmol/2 h (in 2 h) Productivity ratio

acriflavin + dehydrogenase/dehydrogenase

Lactic acid LDH 200 Ethanol AIDH 306 Malic acid MDH 83 Alanine AlaDH 112 Glutamic acid GluDH(NAD ÷) 79 Glutamic acid GluDH (NADP ÷) 51

2.0 (40) a 9.6 (225) a 3.1 5.6 0.8 4.6 1.1 7.5 0.8 1.8 0.5 1.3

a Il lumination for 80 h


OH I

HO2C-CH-CH2CO2H + 1/202-~

0 II

HO2C-C-CH2CO2H + H20


rosion to poisonous materials, i.e. CdS semiconductor powders. ~06

The success in developing photochemical regeneration systems of NAD(P)+-NAD(P)H cofactors sug- gests that other energy-requiring cofactors could be

(47) recycled by similar routes, i.e. ATP regeneration. The possibilities of coupling enzymes directly to light-derived electron carriers (redox mediators), i.e. reduction of NO~- or CO2, seems attractive for practical application and rapid progress using this approach can be anticipated.

For practical applications of NAD(P)+-NAD(P)H - dependent biotransformation (including photochemical regeneration), immobilization of the expensive cofactors is essential, Such immobilization requires communication capabilities of the cofactors with other biocatalysts or catalysts in the reactor configuration. In photochemical systems, immobilization of the light- active compounds, cofactors, redox mediators, catalysts, or biocatalysts in a communicating assembly will be essential for practical applications. The impressive demonstration by Wichman e t a l . , j°7 who linked NAD ÷ to polyethylene glycol and used the immobilized cofactor in an enzyme reactor for the synthesis of L-leucine, could be applied also to photochemical NAD(P)+-NAD(P)H regeneration assemblies. Orga- nization of colloidal semiconductor particles or poly- mer-anchored photosensitizers 1°8-~° with immobilized NAD(P) + cofactors and appropriate enzymes in hollow fiber or membrane-based configurations seem

(49) to be future challenges for research.

HO2C-CH2-CH2CHCO2H + 1/202"~ I

NH2

O LL

HO2C-CH2-CH2-C-CO2H + H20 (48)

With methylene blue (MB +) or N-methyl-phenazonium methyl sulfate (MPMS+), which act as photosensitizers for the regeneration of NADP +, the enzymatic oxidation of 6-phosphate-gluconate has been accomplished. 87 In this process, (equation 49), oxidation of gluconate-6-phosphate to ribulose-5-phosphate and carbon dioxide occurs. The process is catalysed by NADP+-dependent 6-phosphogluconate dehydrogenase (6-PGDH) and NADP + is regenerated through the photocatalysed oxidation of NADPH by oxygen.

6-P-gluconate + NADP + ~PODH ) hu

ribulose-5-P + CO2 + NADPH + H +

Conclus ions and prospects

Extensive progress in the design of biotransformations through coupling of visible solar light energy and biocatalysts has been accomplished in recent years. Application of light energy is attractive for processes that require the supply of energy as a driving force, i.e. endoergic reaction. The regeneration of NAD(P)+/ NAD(P)H cofactors by photochemical means provides numerous possibilities to be applied in oxidative or reductive biocatalytic processes. The survey reveals that several processes have applied artificial redox mediators that effect NAD(P) ÷ and NAD(P)H regeneration without the aid of natural enzymes. This is certainly of importance to increase the stabilities of the systems in subsequent cofactor-dependent biotransformations. Nevertheless, the photochemical approaches are not free of limitations: solar light is a diffuse energy source and photon harnessing and concomitant heat effects might be severe difficulties in practical applicability. Yet, the combination of biocatalysts and light might be a challenging attitude in production of valuable chemicals, i.e. food products or fuel in arid areas.

The photocatalysts included in such NAD(P)+/ NAD(P)H regeneration systems are not free of limitations. While their synthesis is straightforward and large quantities can be prepared inexpensively, some are photochemically degraded or undergo photocor-

A c k n o w l e d g e m e n t

The support of our studies by the Belfer Foundation and Singer Fund is gratefully acknowledged.

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enzyme-catalysed biotransformations through photochemical regeneration of nicotinamide cofactors

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