Molecular Basis for Thermoprotection in Bemisia: Structural Differences Between Whiteﬂy Ketose Reductase and Other Medium- Chain Dehydrogenases/Reductasesଝ Gregory R

Insect Biochemistry and Molecular Biology 29 (1999) 113–120

Molecular basis for thermoprotection in Bemisia: structural differences between whiteﬂy ketose reductase and other medium- chain dehydrogenases/reductasesଝ Gregory R. Wolfe a, Clyde A. Smith b, Donald L. Hendrix a, Michael E. Salvucci a,* a Western Cotton Research Laboratory, US Department of Agriculture, Agricultural Research Service, Phoenix, AZ 85040, USA b School of Biological Sciences, University of Auckland, Auckland, New Zealand

Received 21 July 1998; received in revised form 13 October 1998; accepted 14 October 1998

Abstract

The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) accumulates sorbitol as a thermoprotectant in response to elevated temperature. Sorbitol synthesis in this insect is catalyzed by an unconventional ketose reductase (KR) that uses NADPH to reduce fructose. A cDNA encoding the NADPH-KR from adult B. argentifolii was cloned and sequenced to determine the primary structure of this enzyme. The cDNA encoded a protein of 352 amino acids with a calculated molecular mass of 38.2 kDa. The deduced amino acid sequence of the cDNA shared 60% identity with sheep NAD+-dependent sorbitol dehydrogenase (SDH). Residues in SDH involved in substrate binding were conserved in the whitefly NADPH-KR. An important structural difference between the whitefly NADPH-KR and NAD+-SDHs occurred in the nucleotide-binding site. The Asp residue that coordinates the adenosyl ribose hydroxyls in NAD+-dependent dehydrogenases (including NAD+-SDH), was replaced by an Ala in the whitefly NADPH-KR. The whitefly NADPH-KR also contained two neutral to Arg substitutions within four residues of the Asp to Ala substitution. Molecular modeling indicated that addition of the Arg residues and loss of the Asp decreased the electric potential of the adenosine ribose-binding pocket, creating an environment favorable for NADPH-binding. Because of the ability to use NADPH, the whitefly NADPH-KR synthesizes sorbitol under physiological conditions, unlike NAD+-SDHs, which function in sorbitol catabolism.  1999 Elsevier Science Ltd. All rights reserved.

Keywords: Sorbitol dehydrogenase; Polyols; Heat stress; Insect carbohydrates

1. Introduction al., 1997). However, evidence that organisms accumulate polyols as a mechanism to mitigate the effects of Many organisms accumulate polyhydric alcohols heat stress was only recently presented (Wolfe et al., (polyols) for protection against osmotic (Yancey et al., 1998; Hendrix and Salvucci, 1998). These studies 1982) and chilling stress (Lee et al., 1987; Storey and showed that two groups of insects, whiteflies and aphids, Storey, 1992), and to prevent freezing (Storey and Sto- accumulate sorbitol and mannitol, respectively, in rey, 1992). Polyols also protect against high temperature response to heat stress (Wolfe et al., 1998; Hendrix and damage when added exogenously to cell cultures (Henle Salvucci, 1998). and Warters, 1982; Kim and Lee, 1993) and to isolated The conventional pathway for sorbitol biosynthesis in enzymes (Black et al., 1979; Erarslan, 1995; Wimmer et most animals, including insects, is via the reduction of glucose catalyzed by NADPH-dependent aldose reductase (EC 1.1.1.21) (Storey and Storey, 1992; Jeff- Abbreviations: KR, ketose reductase; NADPH-KR, NADPH-depen- ery and Jo¨rnvall, 1988). Catabolism of sorbitol in ani- dent ketose reductase; NAD+-SDH, NAD+-dependent sorbitol dehydro- mals involves an NAD+-dependent dehydrogenase genase; SDH, sorbitol dehydrogenase. (SDH; EC 1.1.1.14) that oxidizes sorbitol to fructose ଝ The nucleotide sequence reported in this paper has been submitted to the Genbank/NCBI Data bank with Accession Number AF067126. (Storey and Storey, 1992; Jeffery and Jo¨rnvall, 1988). * Corresponding author. Tel.: +1-602-379-3524; fax: +1-602-379- In a previous study (Wolfe et al., 1998), we identified an 4509; e-mail: [email protected] enzyme in the silverleaf whitefly (Bemisia argentifolii,

Bellows and Perring) that catalyzes the synthesis of sor- 2.2. cDNA library construction bitol from fructose. This enzyme has a strict requirement for NADP(H) in contrast to the mammalian SDHs that Adult silverleaf whiteflies were collected from cotton are specific for NAD(H) (Jeffery and Jo¨rnvall, 1988). plants (Gossypium hirsutum L.) in a greenhouse Purification of the whitefly ketose reductase showed that (Salvucci et al., 1997). Total whitefly RNA was isolated the enzyme is a homotetramer composed of 38.7 kDa using Trizol reagent (Gibco BRL, Grand Island, NY)1 subunits (Salvucci et al., 1998). The amino acid following the manufacturer’s instructions. PolyA-RNA sequences of peptides from the whitefly protein were was isolated from total RNA using Oligo dT Dynabeads homologous to mammalian SDHs. Thus, whiteflies con- (Dynal A.S., Oslo, Norway). A cDNA library was pre- tain an NADPH-dependent ketose reductase (NADPH- pared using ZAP Express cDNA Synthesis Kit and ZAP KR; EC 1.1.1.?) that is similar in structure to NAD+- Express cDNA Gigapack III Gold Cloning Kit SDHs but is anabolic and uses NADPH as the coenzyme. (Stratagene, La Jolla, CA). All growth conditions and SDHs are members of the medium-chain de- host strains were as specified by the manufacturer. hydrogenase/reductase (MDR) family (Jeffery and Jo¨rnvall, 1988; Jo¨rnvall et al., 1984). This family 2.3. Mass excision and immunoscreening of the cDNA includes numerous zinc-containing enzymes such as the library well-studied liver alcohol dehydrogenase (ADH; EC 1.1.1.1) (Jo¨rnvall et al., 1984). Like alcohol dehydrogen- The ZAP Express cDNA library was treated with the ases, SDHs catalyze the NAD(H)-dependent interconver- ExAssist helper phage to excise the cDNAs in pBK- sion of alcohols with their corresponding ketones or CMV phagemids as described by the supplier aldehydes. A model of the structure of sheep liver SDH (Stratagene). Excised phagemids were transformed into was constructed (Eklund et al., 1985) based upon amino E. coli XL1-Blue competent cells (Sambrook et al., acid sequence alignment and the crystallographically- 1989) and transformants were selected by growing col- determined tertiary structure of horse liver ADH (Eklund onies on LB agar plates (150 mm diameter) containing et al., 1976, 1981). The model shows that the coenzyme- 50 µgmlϪ1 kanamycin overnight at 37°C. Colonies were binding domains of ADH and SDH are conserved, even transferred to LB/kanamycin agar plates containing 0.17 though these enzymes differ in substrate specificity. Both mM isopropylthio-β-D-galactoside (IPTG) using sterile ADH and SDH contain a catalytic zinc atom in their nitrocellulose membranes and incubated at 37°C for 3 active sites. However, ADH also possesses a second, h. Immunological screening of the colonies was carried structural zinc atom (Eklund et al., 1981) that is not out as described (Sambrook et al., 1989) using poly- present in SDH (Jeffery et al., 1984b). The structural clonal antibodies raised in rabbits against whitefly zinc atom and the surrounding amino acids are involved NADPH-KR (Salvucci et al., 1998). in maintaining stability of ADH and may be important for subunit association in the quaternary structure 2.4. DNA sequencing and analysis of the deduced (Eklund et al., 1985; Karlsson et al., 1995). The func- amino acid sequence tional form of NAD+-ADH is homodimeric, in contrast to NAD+-SDH which is a homotetramer. The nucleotide sequence of the cDNA clone for the Here we cloned and sequenced the whitefly NADPH- whitefly NADPH-KR was determined by fluorescence KR to compare its primary structure with that of NAD+- autosequencing of both strands using an Applied Biosys- SDH. The results elucidate the structural basis for the tems model 377 automated sequencer located at the Ari- altered coenzyme requirement of the whitefly enzyme. zona State University Sequencing Laboratory. The deduced amino acid sequence of the whitefly NADPH- KR/SDH was aligned with previously published SDH sequences using the Multiple Sequence Alignment pro- 2. Materials and methods gram originally developed by the National Center of Biotechnological Information (Lipman et al., 1989). Alignments of SDH with ADH were based on previously 2.1. Feeding experiments published alignment patterns (Jeffery and Jo¨rnvall, 1988; Eklund et al., 1985). Whiteflies were supplied for a total of 4 h with artificial diets containing 20% sucrose (Salvucci et al., 1997). The temperature of the feeders was increased by 5°C every 30 min until the indicated temperature was 1 Mention of a trademark, proprietary product, or vendor does not achieved. Sorbitol content was determined by anion- constitute a guarantee or warranty of the product by the US Department exchange HPLC as described previously (Wolfe et al., of Agriculture and does not imply its approval to the exclusion of other 1998). products or vendors that may also be suitable. G.R. Wolfe et al./Insect Biochemistry and Molecular Biology 29 (1999) 113–120 115

2.5. Analysis of expressed NADPH-KR and KR ranging from 25 to 41°C while feeding on artificial diets activity containing 20% sucrose. Analysis of whitefly carbohydrates and polyols showed that sorbitol content E. coli XL1-Blue cells harboring the whitefly increased in a temperature dependent manner through NADPH-KR phagemid clone were grown overnight at the range of temperatures examined (Table 1). The 37°C in terrific broth (Sambrook et al., 1989) sup- amount of sorbitol in whiteflies at 41°C was ten-fold plemented with 50 µgmlϪ1 kanamycin. The overnight higher than at 25°C, and equivalent to a hemolymph con- culture was diluted five-fold with fresh medium and, centration of approximately 400 mM (see Wolfe et al., after1hat37°C, was supplemented with 0.42 mM IPTG 1998). We showed previously that synthesis of sorbitol to induce synthesis of recombinant protein. After 3 h, in whiteflies is catalyzed by an unusual NADPH-requir- the cells were harvested by centrifugation for 10 min at ing ketose reductase (Wolfe et al., 1998; Salvucci et 4000g and the cell pellets were suspended in 50 mM al., 1998). HEPES-KOH (pH 7.9). Cells were lysed by sonication To isolate a cDNA encoding NADPH-KR a whitefly at 4°C and the cell lysates were subjected to immunoblot cDNA expression library was prepared and screened analysis and assayed for NADPH-KR activity (Salvucci with polyclonal antibodies directed against whitefly et al., 1998). For immunoblot analysis, cell lysates con- NADPH-KR. One immunoreactive clone was obtained taining equal amounts of protein were electrophoresed from screening approximately 2.4 × 104 colonies. The on 11% SDS-PAGE gels (Salvucci et al., 1998). Poly- isolated phagemid from this clone contained a 2005 bp peptides were transferred from the gels to Immobilon-P cDNA insert. PVDF membranes (Millipore Corp., Bedford, MA) as To verify that the 2005 bp cDNA clone encoded whit- described (Salvucci et al., 1998). Blots were probed with efly NADPH-KR, the expressed protein was analyzed on antibodies against whitefly NADPH-KR and then with Western blots and assayed for activity (Fig. 1). Anti- an alkaline phosphatase-conjugated secondary antibody bodies directed against whitefly NADPH-KR recognized (Salvucci et al., 1993). Recombinant NADPH-KR was a polypeptide on Western blots of extracts of E. coli cells isolated from cell lysates as described (Salvucci et al., harboring the positive clone, but not in extracts contain- 1998). ing a non-reactive clone (i.e., a randomly selected phagemid-containing colony that was not immunoreactive dur- 2.6. Molecular modeling ing library screening). The reactive band had an apparent molecular weight of 39 kDa, similar in size to the pur- Models for the 3-dimensional structure of insect ified whitefly NADPH-KR. Assays of NADPH-depen- ketose reductase were constructed from the theoretically- dent fructose reduction showed that extracts prepared derived structure of the sheep liver SDH/NAD+ complex from the immunologically positive clone also contained (Eklund et al., 1985) and the refined structure of horse catalytically-active NADPH-KR. In contrast, no liver ADH/NAD+ complex (Eklund et al., 1981) (PDB NADPH-KR activity was detected in extracts from the accession codes 1SDG and 6ADH). The region 199–206, non-reactive E. coli transformant. After purification, the implicated in adenine–ribose binding in NAD+-SDH specific activity of the recombinant enzyme was virtually (VTDLSASR), and 221–228 in ADH (GVDINKDK) identical to the enzyme purified from whiteflies were mutated to the corresponding residues (197–204) (Salvucci et al., 1998). Also like the whitefly enzyme, in NADPH-KR (CTARSPRR) based upon a multiple the recombinant enzyme displayed an almost exclusive sequence alignment. The mutated residues were built in preference for NADPH. Taken together, these results their most favored rotamer conformation. Both models demonstrate that the 2005 bp cDNA insert encodes the showed essentially the same features with respect to whitefly NADPH-KR. cofactor binding. The model-building program TURBO The nucleotide sequence of the coding strand for whit- (Roussel, A., Inisan, A.-G. and Cambillau, C., AFMB efly NADPH-KR and the deduced amino acid sequence and Biographics, Marseille, France) running on a SGI Indigo2 was used for all modeling and structural analysis. Table 1 Effect of temperature on sorbitol accumulation in whitefly bodies

Temperature (°C) Sorbitol content (nmol 3. Results whiteﬂy-1)

Previous studies showed that sorbitol accumulates in 25 0.14 ± 0.06 the bodies of whiteﬂies when these insects are shifted 30 0.24 ± 0.05 ° 34 0.28 ± 0.02 from 25 to 41 C (Wolfe et al., 1998). To document the 37 0.72 ± 0.14 temperature dependence of polyol accumulation in this 41 1.38 ± 0.70 insect, adult whiteﬂies were exposed to temperatures 116 G.R. Wolfe et al./Insect Biochemistry and Molecular Biology 29 (1999) 113–120

sequenced (Jo¨rnvall et al., 1984, 1987) were also present in the whitefly NADPH-KR (Fig. 3). Residues implicated in sorbitol binding in sheep liver SDH (Eklund et al., 1985), including Cys-41, Ser-43, Tyr-47, Ile-53, His-66, Pro-92, Phe-115, Pro-120, Glu- 152, Ile-191 and Lys-192 were conserved in NADPH- KR. In addition, the four-cysteine motif that binds the structural zinc in ADHs, was also conserved in the whitefly NADPH-KR (i.e., Cys-96, Cys-99, Cys-102, Cys- 110 of the whitefly enzyme). All four of these residues were also present in the B. mori SDH, but not in mammalian SDHs. Several of the residues implicated in NAD+-binding by the sheep liver SDH, including Ser-48, His-51, Arg- 228 and Lys-369 (Eklund et al., 1985) were conserved in the whitefly NADPH-KR. A noteworthy difference between the whitefly NADPH-KR and the NAD+-SDH and -ADH sequences was the absence in the whitefly enzyme of the conserved Asp that hydrogen bonds with the 2Ј and 3Ј-hydroxyls of the adenosyl ribose of NAD+. In place of this Asp, the whitefly NADPH-KR has an Ala residue. Another potentially important difference was the Fig. 1. Expression of the cDNA for whitefly NADPH-KR in E. coli. presence of Arg residues at positions 200 and 203 of the Immunoblot analysis of (1) NADPH-KR purified from whitefly, (2) whitefly enzyme. These Arg residues replaced uncharged total protein from E. coli transformed with a phagemid lacking the residues in NAD+-SDH and an uncharged and a nega- NADPH-KR cDNA, (3) total protein from E. coli transformed with a phagemid containing the NADPH-KR cDNA insert. Lanes 2 and 3 tively charged residue in horse liver ADH. were loaded on an equal protein basis. The numbers below the lanes Molecular modeling was used to compare the struc- show the activity of NADPH-KR in each sample. tures of the coenzyme-binding sites of NADPH-KR and NAD+-SDH. The adenosyl-ribose-binding domain of NAD+-SDH (199VTDLSASR) and ADH (221GVDIN- are presented in Fig. 2. The cDNA contained an open KDK) was “mutated” to the corresponding residues of reading frame of 1056 bp that encoded a polypeptide of NADPH-KR (197CTARSPRR). Calculation of the elec- 352 amino acids with a calculated molecular mass of trostatic surface inside the binding pockets of the native 38,164 Da. The open reading frame was bounded by a and mutated enzymes showed that in the former case, 102 bp 5Ј-untranslated region and a TAA termination the presence of the conserved Asp produced a negative codon at the 3Ј end. The coding region was followed by charge inside the cavity. In the enzyme “mutated” to the complete 3Ј-untranslated region, 847 bases terminat- resemble NADPH-KR, this cavity was positively ing in a poly(A) tail. Though a classical AATAAA poly- charged due primarily to the replacement of the con- adenylation signal was not found, a putative polyadenyl- served Asp with Ala and the replacement of the adjacent ation signal, TATAAA, was located 17 bases upstream Leu or Ile with Arg. Consequently, both sides of the of the poly(A) tail. The large 3Ј-untranslated sequence pocket in the “mutated” enzyme were lined by positive contained 20 in-frame termination codons. Three charges, namely Arg-200, added by mutation, and the stretches of the deduced sequence, underlined in Fig. 2, conserved Arg, Arg-204 (Fig. 4). The model showed that matched the amino acid sequences obtained from tryptic a second Arg residue added by mutation, Arg-203 in the peptide and cyanogen bromide fragments of purified NADPH-KR/SDH, was at the surface of the molecule whitefly NADPH-KR (Salvucci et al., 1998). approximately 9 A˚ from the phosphate of NADP, and The amino acid sequences of whitefly NADPH-KR did not appear to influence cofactor binding. The model were aligned with NAD+-SDHs and ADH (Fig. 3). Com- also showed that Ser-201 in the NADPH-KR may inter- parison of the sequences showed that the whitefly act with the phosphate of NADPH, further stabilizing enzyme and the sheep liver SDH (Jeffery et al., 1984a) the complex. were 60% identical at the amino acid level. The whitefly enzyme exhibited 41% identity with SDH from Bombyx mori (Niimi et al., 1993, 1996) and 21% with horse liver 4. Discussion ADH (Eklund et al., 1985; Jo¨rnvall, 1970). Nineteen of the twenty-two amino acids that are conserved in most Feeding experiments demonstrated that accumulation of the NAD+-ADHs and NAD+-SDHs that have been of sorbitol in the whitefly is not solely a heat shock G.R. Wolfe et al./Insect Biochemistry and Molecular Biology 29 (1999) 113–120 117

Fig. 2. Nucleotide and deduced amino acid sequence of the cDNA encoding the whitefly NADPH-KR. The underlined regions correspond to the sequences obtained from amino acid sequencing of tryptic and cyanogen bromide peptides from the purified protein (Salvucci et al., 1998). The termination codon is indicated by bold underlined type and the putative polyadenylation signal is indicated in bold type. response, but increases through a range of temperatures tures from hyperthermal killing (Henle and Warters, from 25 to 41°C. In this study and previously (Wolfe et 1982) and to increase thermal stability of isolated al., 1998), sorbitol concentrations exceeded 400 mM in enzymes (Black et al., 1979; Erarslan, 1995; Wimmer et whitefly hemolymph when whiteflies were exposed to al., 1997). elevated temperatures while feeding on artificial diets Comparison of the deduced amino acid sequence of containing sucrose. This polyol concentration exceeds whitefly NADPH-KR with sequences available in the concentrations required to protect mammalian cell cul- database confirmed its relatedness to NAD+-dependent 118 G.R. Wolfe et al./Insect Biochemistry and Molecular Biology 29 (1999) 113–120

Fig. 3. Alignment of the deduced amino acid sequences of whitefly NADPH-KR with sequences of B. mori NAD+-SDH (Bm SDH), sheep NAD+- SDH (S SDH) and horse liver ADH (H ADH). Amino acid identities, compared to the whitefly sequence, are highlighted and boxed. Residues generally conserved throughout the MDR family (Jeffery and Jo¨rnvall, 1988) are indicated by a (¼) above. Putative zinc-coordinating residues are subtended by a z (catalytic zinc) or by a z (structural zinc). Amino acids putatively responsible for the altered specificity of the coenzyme-binding site of NADPH-KR are indicated by white letters in a black field.

Fig. 4. Structural comparison of the nucleotide binding pocket in models of the (A) sheep liver NAD+-SDH (Eklund et al., 1985) and (B) whiteﬂy NADPH-KR. The region of NAD+-SDH from 199–206 (VTDLSASR) was mutated to the corresponding residues in NADPH-KR (CTARSPRR). The NAD+ and NADP+ are shown for SDH and KR respectively. The structures were generated by MOLSCRIPT (Kraulis, 1991). G.R. Wolfe et al./Insect Biochemistry and Molecular Biology 29 (1999) 113–120 119

SDH. SDHs are members of the MDR superfamily that involved in zinc binding, while the nicotinamide ring is include ADHs, ζ-crystallin (Borras et al., 1989) and buried in a hydrophobic pocket within the molecule. All quinone oxidoreductases (Persson et al., 1994). the regions involved in cofactor binding in NAD+-SDH Throughout this family, only 22 amino acid residues are and NAD+-ADH are conserved in NADPH-KR with the highly conserved (Jeffery and Jo¨rnvall, 1988). Nineteen notable exception of the region 197–203, the putative of the 22 residues were conserved in the NADPH-KR NADH/NADPH specificity pocket. (Fig. 3). The exceptions were an invariant Asp that was The structural model of sheep liver SDH showed that replaced with an Ala in the whitefly NADPH-KR (i.e., Asp-201 forms hydrogen bonds with the 2Ј- and 3Ј- Ala-199) and a Gly that was replaced with Ser at pos- hydroxyl groups of the adenosyl ribose of NAD(H) ition 293 of the whitefly enzyme. (Eklund et al., 1985). This Asp was not conserved in the Although SDH and ADH are both zinc-containing NADPH-KR. The presence or absence of an Asp residue enzymes, ADHs contain two zinc atoms per subunit, one in coenzyme binding sites in other families of dehydro- structural and the other catalytic, while SDH has only a genases determines the specificity for NAD+ or NADP+ single catalytic zinc atom (Jeffery et al., 1984b). In the (Scrutton et al., 1990; Chen et al., 1991; Bocanegra et crystallographic structure of the horse liver ADH, Cys- al., 1993). Molecular modeling of this region showed 46, His-67 and Cys-174 coordinate the catalytic zinc that the amino acid substitutions in the whitefly (Eklund et al., 1976, 1981). Cys-46 and His-67 are con- NADPH-KR decrease the electronegativity of the aden- served in SDH (Jeffery et al., 1984b) and Glu-150 has osine binding pocket, thereby providing an environment been implicated as the third member of the zinc coordin- favorable for NADPH binding. The model shows that ating triad of SDH based on site-directed mutagenesis the invariant Arg at position 206 of the sheep enzyme (Karlsson et al., 1995; Karlsson and Hoö¨g, 1993). All and Ser-203 could potentially hydrogen bond with aden- three of these residues are conserved in NADPH-KR. osyl ribose-phosphate. Interestingly, the structure of the Four Cys residues, Cys-97, Cys-100, Cys-103 and NADP+-dependent ADH from Clostridium beijerinckii Cys-111, are responsible for coordinating the structural (Reid and Fewson, 1994), though displaying relatively zinc atom in NAD+-ADH (Eklund et al., 1976, 1981; low overall amino acid sequence homology to NAD+- Karlsson et al., 1995). Of these four residues, only Cys- SDH or NADPH-KR/SDH, also has a positively charged 103 is conserved in mammalian NAD+-SDH, an obser- adenine-ribose pocket, with Gly in place of the Asp. Like vation consistent with the absence of the structural zinc NADPH-KR, NADPH-ADH has a Ser and an Arg resi- in NAD+-SDH. Interestingly, all four of these Cys residue in this region that could serve as ligands to NADPH dues are conserved in the whitefly NADPH-KR and in (Reid and Fewson, 1994). NAD+-SDH from another insect, B. mori (Fig. 2). Con- servation of the four Cys zinc-binding motif was also reported in the NAD+-SDH from Bacillus subtillus; how- 5. Conclusions ever, analysis by atomic absorption spectroscopy revealed that this enzyme contained only a single zinc The silverleaf whitefly accumulates sorbitol in atom per subunit (Ng et al., 1992). The stoichiometry of response to heat stress (Wolfe et al., 1998). Here we zinc atoms per subunit has not been determined for the showed that sorbitol accumulation in the whitefly body B. mori NAD+-SDH or for the whitefly NADPH-KR and was not simply a heat shock response, but was fully tem- thus the significance of the conservation of the second perature dependent. The primary structure of the enzyme zinc-binding motif in the enzymes from these two insect that synthesizes sorbitol in whiteflies, NADPH-KR, dis- species remains unclear. played high homology to mammalian NAD+-SDH. This In NAD+-SDH (Eklund et al., 1985) and ADH enzyme is the first example of a SDH that prefers (Eklund et al., 1976, 1981), the NAD(H) cofactor-bind- NADP(H). Structural differences in the nucleotide-binding site consists of a groove on the surface of the mol- ing domains between NAD+-SDH and NADPH-KR, ecule with a deep cavity at one end, directed towards particularly replacements of an Asp with an Ala and the the center of the molecule. The adenine, ribose and pyro- adjacent Leu with an Arg, change the electric potential phosphate groups lie in this groove, exposed to the exter- of the nucleotide-binding pocket. Similar replacements nal solvent, while the nicotinamide moiety is buried in have been engineered in other dehydrogenases to alter the cavity. The area adjacent to the adenosyl ribose oxy- the coenzyme preference between NAD(H) and gen atoms, formed from the region of NAD+-SDH NADP(H) (Scrutton et al., 1990; Chen et al., 1991; sequence VTDLSAS (residues 199–205), lies above a Bocanegra et al., 1993). By changing the coenzyme classical Rossmann dinucleotide-binding fold with the requirement of the normally catabolic SDH, whiteflies sequence GAGPIG (residues 177–182), and interacts have apparently evolved an unusual enzyme that func- with the pyrophosphate group. The nicotinamide-ribose tions in sorbitol synthesis. Selection of this enzyme con- interacts with the loop CGSDVH of NAD+-SDH fers survival advantage to whiteflies in hot, arid environ- (residues 44–49). The cysteine residue of this loop is ments since it allows them to convert the copious 120 G.R. Wolfe et al./Insect Biochemistry and Molecular Biology 29 (1999) 113–120 amounts of fructose available from their diet to sorbitol, directed mutagenesis. 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