Structural and biochemical characterization of the nucleoside hydrolase from C. elegans reveals the role of two active site cysteine residues in catalysis

Nucleoside hydrolases (NHs) catalyze the hydrolysis of the N‐glycoside bond in ribonucleosides and are found in all three domains of life. Although in parasitic protozoa a role in purine salvage has been well established, their precise function in bacteria and higher eukaryotes is still largely unknown. NHs have been classified into three homology groups based on the conservation of active site residues. While many structures are available of representatives of group I and II, structural information for group III NHs is lacking. Here, we report the first crystal structure of a purine‐specific nucleoside hydrolase belonging to homology group III from the nematode Caenorhabditis elegans (CeNH) to 1.65Å resolution. In contrast to dimeric purine‐specific NHs from group II, CeNH is a homotetramer. A cysteine residue that characterizes group III NHs (Cys253) structurally aligns with the catalytic histidine and tryptophan residues of group I and group II enzymes, respectively. Moreover, a second cysteine (Cys42) points into the active site of CeNH. Substrate docking shows that both cysteine residues are appropriately positioned to interact with the purine ring. Site‐directed mutagenesis and kinetic analysis proposes a catalytic role for both cysteines residues, with Cys253 playing the most prominent role in leaving group activation.     

Leaving group activation by aromatic stacking: an alternative to general acid catalysis

General acid catalysis is a powerful and widely used strategy in enzymatic nucleophilic displacement reactions. For example, hydrolysis/phosphorolysis of the N-glycosidic bond in nucleosides and nucleotides commonly involves the protonation of the leaving nucleobase concomitant with nucleophilic attack. However, in the nucleoside hydrolase of the parasite Trypanosoma vivax, crystallographic and mutagenesis studies failed to identify a general acid. This enzyme binds the purine base of the substrate between the aromatic side-chains of Trp83 and Trp260. Here, we show via quantum chemical calculations that face-to-face stacking can raise the pKa of a heterocyclic aromatic compound by several units. Site-directed mutagenesis combined with substrate engineering demonstrates that Trp260 catalyzes the cleavage of the glycosidic bond by promoting the protonation of the purine base at N-7, hence functioning as an alternative to general acid catalysis.     

Crystal structures of T. vivax nucleoside hydrolase in complex with new potent and specific inhibitors

Diseases caused by parasitic protozoa remain a major health problem, mainly due to old toxic drugs and rising drug resistance. Nucleoside hydrolases are key enzymes of the purine salvage pathway of parasites from the Trypanosomatidae family and are considered as possible drug targets. N-Arylmethyl substituted iminoribitols have been developed as selective nanomolar affinity inhibitors against the purine-specific nucleoside hydrolase of Trypanosoma vivax. The current paper describes the crystal structures of the T. vivax nucleoside hydrolase in complex with two of these inhibitors, to 1.3 and 1.85 Å resolution. These high resolution structures provide an accurate picture of the mode of binding of these inhibitors and their mechanism of transition-state mimicry, and are valuable tools to guide further inhibitor design. Comparison of the current structures with previously solved structures of the enzyme in complex with ground-state and transition-state-analogue inhibitors also allows for the elucidation of a detailed molecular mechanism of active-site loop opening/closing. These loop movements can be coupled to the complex kinetic mechanism of the T. vivax nucleoside hydrolase.     

A nonspecific nucleoside hydrolase from Leishmania donovani: implications for purine salvage by the parasite.

In contrast to their mammalian hosts, protozoan parasites do not synthesize purines de novo, but depend on preformed nucleotides that they purportedly obtain by salvage pathways. Nucleoside hydrolases may play a crucial role in that salvage process. By screening Leishmania donovani libraries with polyclonal antibodies against promastigote soluble exo-antigens, we have identified a cDNA encoding a protein with significant homology to nonspecific and uridine–inosine-preferring nucleoside hydrolases. Sequence comparison demonstrated that all the residues involved in Ca²⁺-binding and substrate recognition in the active site are conserved among the characterized protozoan nucleoside hydrolases. Genomic analysis suggests that it is a single copy gene in L. donovani, and its homologues are present in members representing other Leishmania species complexes. Both Northern blot and immunoblot analyses indicate that it is constitutively expressed in L. donovani promastigotes. The recombinant enzyme overexpressed in and purified from bacteria showed significant activity with all naturally occurring purine and pyrimidine nucleosides, and efficient utilization of p-nitrophenyl-β- -ribofuranoside as a substrate. Altogether, the sequence comparison and substrate specificity data identify this L. donovani nucleoside hydrolase as a nonspecific nucleoside hydrolase. Further, the nucleoside hydrolase was localized to specific foci in L. donovani promastigotes by immunofluorescent assays. Although the conservation of the nucleoside hydrolases among protozoan parasites offers promise for the design of broad-spectrum anti-parasitic drugs, the existence of multiple and distinct nucleoside hydrolases in a single species demands special consideration.     

Structural basis for branching-enzyme activity of glycoside hydrolase family 57: structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1

Branching enzymes (BEs) catalyze the formation of branch points in glycogen and amylopectin by cleavage of α-1,4 glycosidic bonds and subsequent transfer to a new α-1,6 position. BEs generally belong to glycoside hydrolase family 13 (GH13); however TK1436, isolated from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, is the first GH57 member, which possesses BE activity. To date, the only BE structure that had been determined is a GH13-type from Escherichia coli. Herein, we have determined the crystal structure of TK1436 in the native state and in complex with glucose and substrate mimetics that permitted mapping of the substrate-binding channel and identification of key residues for glucanotransferase activity. Its structure encompasses a distorted (β/α)(7)-barrel juxtaposed to a C-terminal α-helical domain, which also participates in the formation of the active-site cleft. The active site comprises two acidic catalytic residues (Glu183 and Asp354), the polarizer His10, aromatic gate-keepers (Trp28, Trp270, Trp407, and Trp416) and the residue Tyr233, which is fully conserved among GH13- and GH57-type BEs. Despite TK1436 displaying a completely different fold and domain organization when compared to E. coli BE, they share the same structural determinants for BE activity. Structural comparison with AmyC, a GH57 α-amylase devoid of BE activity, revealed that the catalytic loop involved in substrate recognition and binding, is shortened in AmyC structure and it has been addressed as a key feature for its inability for glucanotransferase activity. The oligomerization has also been pointed out as a possible determinant for functional differentiation among GH57 members.     

The crystal structure of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase 1 represents a conformational intermediate in the reductive activation mechanism of the tetrameric enzyme

Toxoplasma gondii nucleoside triphosphate diphosphohydrolase (NTPDase) 1 was crystallized in an intermediate tetrameric conformation. The crystal structure is similar to that of T. gondii NTPDase3 and represents an inactive conformation as the activating disulfide bridge is not reduced and the active site cleft between the two domains of each monomer is open. However, the arrangement of the monomers within the tetramer differs from that of the inactive form of NTPDase3 and may represent an intermediate conformation on the path of the closure motion of the tetramer induced upon activation. Proteins 2013; 81:1271–1276. © 2013 Wiley Periodicals, Inc.     

Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.     

Pre-steady-state analysis of the nucleoside hydrolase of Trypanosoma vivax. Evidence for half-of-the-sites reactivity and rate-limiting product release

The nucleoside hydrolase (NH) of the Trypanosoma vivax parasite catalyzes the hydrolysis of the N-glycosidic bond in ribonucleosides according to the following reaction: beta-purine (or pyrimidine) nucleoside + H(2)O --> purine (pyrimidine) base + ribose. The reaction follows a highly dissociative nucleophilic displacement reaction mechanism with a ribosyl oxocarbenium-like transition state. This paper describes the first pre-steady-state analysis of the conversion of a number of purine nucleosides. The NH exhibits burst kinetics and behaves with half-of-the-sites reactivity. The analysis suggests that the NH of T. vivax follows a complex multistep mechanism in which a common slow step different from the chemical hydrolysis is rate limiting. Stopped-flow fluorescence binding experiments with ribose indicate that a tightly bound enzyme-ribose complex accumulates during the enzymatic hydrolysis of the common purine nucleosides. This is caused by a slow isomerization between a tight and a loose enzyme-ribose complex forming the rate-limiting step on the reaction coordinate.     

Crystal structure of the Bacillus anthracis nucleoside diphosphate kinase and its characterization reveals an enzyme adapted to perform under stress conditions

Nucleoside diphosphate kinases (Ndks) play an important role in a plethora of regulatory and metabolic functions. Inhibition of the B. anthracis Ndk mRNA results in the formation of nonviable aberrant spores. We report the characterization and crystal structure of the enzyme from B. anthracis nucleoside diphosphate kinase (BaNdk), the first from sporulating bacteria. The enzyme, although from a mesophilic source, is active at extremes of pH (3.5–10.5), temperature (10–95°C) and ionic strength (0.25–4.0M NaCl). It exists as a hexamer that is composed of two SDS‐stable trimers interacting in a back‐to‐back association; mutational analysis confirms that the enzyme is a histidine kinase. The high‐resolution crystal structure reported here reveals an unanticipated change in the conformation of residues between 43 and 63 that also regulates substrate entry in other Ndks. A comparative structural analysis involving Ndks from seven mesophiles and three thermophiles has resulted in the delineation of the structure into relatively rigid and flexible regions. The analysis suggests that the larger number of intramolecular hydrogen bonds and to a lesser extent ionic interactions in BaNdk contributes to its high thermostability. Mutational analysis and Molecular Dynamics simulations were used to probe the role of a highly conserved Gly19 (present at the oligomeric interface in most of the Ndks). The results suggest that the mutation leads to a rigidification of those residues that facilitate substrate entry and consequently leads to a large reduction in the kinase activity. Overall, the enzyme characterization helps to understand its apparent adaptation to perform under stress conditions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Structure of the Sulfolobus solfataricus alpha-glucosidase: implications for domain conservation and substrate recognition in GH31

The crystal structure of alpha-glucosidase MalA from Sulfolobus solfataricus has been determined at 2.5Angstrom resolution. It provides a structural model for enzymes representing the major specificity in glycoside hydrolase family 31 (GH31), including alpha-glucosidases from higher organisms, involved in glycogen degradation and glycoprotein processing. The structure of MalA shows clear differences from the only other structure known from GH31, alpha-xylosidase YicI. MalA and YicI share only 23% sequence identity. Although the two enzymes display a similar domain structure and both form hexamers, their structures differ significantly in quaternary organization: MalA is a dimer of trimers, YicI a trimer of dimers. MalA and YicI also differ in their substrate specificities, as shown by kinetic measurements on model chromogenic substrates. In addition, MalA has a clear preference for maltose (Glc-alpha1,4-Glc), whereas YicI prefers isoprimeverose (Xyl-alpha1,6-Glc). The structural origin of this difference occurs in the -1 subsite where MalA residues Asp251 and Trp284 could interact with OH6 of the substrate. The structure of MalA in complex with beta-octyl-glucopyranoside has been determined. It reveals Arg400, Asp87, Trp284, Met321 and Phe327 as invariant residues forming the +1 subsite in the GH31 alpha-glucosidases. Structural comparisons with other GH families suggest that the GH31 enzymes belong to clan GH-D.     

Two fluorogenic substrates for purine nucleoside phosphorylase,selective for mammalian and bacterial forms of the enzyme

Two nontypical nucleosides, 7-β-d-ribosyl-2,6-diamino-8-azapurine and 8-β-d-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λ_max ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λ_max ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.     

Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism.

The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution. The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation). By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation). Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site. This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism. In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation. Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form. The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204. The mechanism explains the broad specificity of E. coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates. The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor.     

The synthesis and X-ray structure of a chiral rhodium-NHC complex: Implications for the use of NHCs in asymmetric hydroformylation catalysis

An optically active N-heterocyclic carbene (NHC) has been metallated with [RhCl(COD)]₂ to give a rhodium(I) monocarbene substituted complex. This complex has been fully characterized by ¹H and ¹³C NMR spectroscopy and by single-crystal X-ray analysis. Preliminary tests of hydroformylation of styrene catalyzed with this compound have shown that branched to linear aldehyde ratio was rather low 0.67 (40%) and that the enantiomeric excess measured on the corresponding alcohols was only 10%. In the presence of an excess of triphenylphosphine, the ratio of branched to linear aldehyde increased markedly (91%) whereas the enantioselectivity of the reaction remained almost unchanged (12.5%).     

Three‐dimensional structure of xylonolactonase from Caulobacter crescentus: A mononuclear iron enzyme of the 6‐bladed β‐propeller hydrolase family

Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d‐xylonolactone. We have determined crystal structures of Cc XylC in complex with d‐xylonolactone isomer analogues d‐xylopyranose and (r)‐(+)‐4‐hydroxy‐2‐pyrrolidinone at high resolution. Cc XylC has a 6‐bladed β‐propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe²⁺. The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side chains of three amino acid residues, Glu18, Asn146, and Asp196, which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe²⁺. The structure of Cc XylC was compared with eight other ester hydrolases of the β‐propeller hydrolase family. The previously published crystal structures of other β‐propeller hydrolases contain either Ca²⁺, Mg²⁺, or Zn²⁺ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe²⁺ as a catalytic metal. This could further expand our understanding of utilization of Fe²⁺ not only in oxidative enzymes but also in hydrolases.     

Structures of N-acetylornithine transcarbamoylase from Xanthomonas campestris complexed with substrates and substrate analogs imply mechanisms for substrate binding and catalysis

N-acetyl-L-ornithine transcarbamoylase (AOTCase) is a new member of the transcarbamoylase superfamily that is essential for arginine biosynthesis in several eubacteria. We report here crystal structures of the binary complexes of AOTCase with its substrates, carbamoyl phosphate (CP) or N-acetyl-L-ornithine (AORN), and the ternary complex with CP and N-acetyl-L-norvaline. Comparison of these structures demonstrates that the substrate-binding mechanism of this novel transcarbamoylase is different from those of aspartate and ornithine transcarbamoylases, both of which show ordered substrate binding with large domain movements. CP and AORN bind to AOTCase independently, and the main conformational change upon substrate binding is ordering of the 80's loop, with a small domain closure around the active site and little movement of the 240's loop. The structures of the complexes provide insight into the mode of substrate binding and the mechanism of the transcarbamoylation reaction.     

Crystal structure of Thermotoga maritima 4-alpha-glucanotransferase and its acarbose complex: implications for substrate specificity and catalysis

4-alpha-Glucanotransferase (GTase) is an essential enzyme in alpha-1,4-glucan metabolism in bacteria and plants. It catalyses the transfer of maltooligosaccharides from an 1,4-alpha-D-glucan molecule to the 4-hydroxyl group of an acceptor sugar molecule. The crystal structures of Thermotoga maritima GTase and its complex with the inhibitor acarbose have been determined at 2.6A and 2.5A resolution, respectively. The GTase structure consists of three domains, an N-terminal domain with the (beta/alpha)(8) barrel topology (domain A), a 65 residue domain, domain B, inserted between strand beta3 and helix alpha6 of the barrel, and a C-terminal domain, domain C, which forms an antiparallel beta-structure. Analysis of the complex of GTase with acarbose has revealed the locations of five sugar-binding subsites (-2 to +3) in the active-site cleft lying between domain B and the C-terminal end of the (beta/alpha)(8) barrel. The structure of GTase closely resembles the family 13 glycoside hydrolases and conservation of key catalytic residues previously identified for this family is consistent with a double-displacement catalytic mechanism for this enzyme. A distinguishing feature of GTase is a pair of tryptophan residues, W131 and W218, which, upon the carbohydrate inhibitor binding, form a remarkable aromatic "clamp" that captures the sugar rings at the acceptor-binding sites +1 and +2. Analysis of the structure of the complex shows that sugar residues occupying subsites from -2 to +2 engage in extensive interactions with the protein, whereas the +3 glucosyl residue makes relatively few contacts with the enzyme. Thus, the structure suggests that four subsites, from -2 to +2, play the dominant role in enzyme-substrate recognition, consistent with the observation that the smallest donor for T.maritima GTase is maltotetraose, the smallest chain transferred is a maltosyl unit and that the smallest residual fragment after transfer is maltose. A close similarity between the structures of GTase and oligo-1,6-glucosidase has allowed the structural features that determine differences in substrate specificity of these two enzymes to be analysed.     

Probing the role of a mobile loop in substrate binding and enzyme activity of human salivary amylase

Mammalian amylases harbor a flexible, glycine-rich loop 304GHGAGGA(310), which becomes ordered upon oligosaccharide binding and moves in toward the substrate. In order to probe the role of this loop in catalysis, a deletion mutant lacking residues 306-310 (Delta306) was generated. Kinetic studies showed that Delta306 exhibited: (1) a reduction (>200-fold) in the specific activity using starch as a substrate; (2) a reduction in k(cat) for maltopentaose and maltoheptaose as substrates; and (3) a twofold increase in K(m) (maltopentaose as substrate) compared to the wild-type (rHSAmy). More cleavage sites were observed for the mutant than for rHSAmy, suggesting that the mutant exhibits additional productive binding modes. Further insight into its role is obtained from the crystal structures of the two enzymes soaked with acarbose, a transition-state analog. Both enzymes modify acarbose upon binding through hydrolysis, condensation or transglycosylation reactions. Electron density corresponding to six and seven fully occupied subsites in the active site of rHSAmy and Delta306, respectively, were observed. Comparison of the crystal structures showed that: (1) the hydrophobic cover provided by the mobile loop for the subsites at the reducing end of the rHSAmy complex is notably absent in the mutant; (2) minimal changes in the protein-ligand interactions around subsites S1 and S1', where the cleavage would occur; (3) a well-positioned water molecule in the mutant provides a hydrogen bond interaction similar to that provided by the His305 in rHSAmy complex; (4) the active site-bound oligosaccharides exhibit minimal conformational differences between the two enzymes. Collectively, while the kinetic data suggest that the mobile loop may be involved in assisting the catalysis during the transition state, crystallographic data suggest that the loop may play a role in the release of the product(s) from the active site.     

B. subtilis ykuD protein at 2.0 A resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes

The crystal structure of the product of the Bacillus subtilis ykuD gene was solved by the multiwavelength anomalous dispersion (MAD) method and refined using data to 2.0 A resolution. The ykuD protein is a representative of a distinctly prokaryotic and ubiquitous family found among both pathogenic and nonpathogenic Gram-positive and Gram-negative bacteria. The deduced amino acid sequence reveals the presence of an N-terminal LysM domain, which occurs among enzymes involved in cell wall metabolism, and a novel, putative catalytic domain with a highly conserved His/Cys-containing motif of hitherto unknown structure. As the wild-type protein did not crystallize, a double mutant was designed (Lys117Ala/Gln118Ala) to reduce excess surface conformational entropy. As expected, the structure of the LysM domain is similar to the NMR structure reported for an analogous domain from Escherichia coli murein transglycosylase MltD. The molecular model also shows that the 112-residue-long C-terminal domain has a novel tertiary fold consisting of a beta-sandwich with two mixed sheets, one containing five strands and the other, six strands. The two beta-sheets form a cradle capped by an alpha-helix. This domain contains a putative catalytic site with a tetrad of invariant His123, Gly124, Cys139, and Arg141. The stereochemistry of this active site shows similarities to peptidotransferases and sortases, and suggests that the enzymes of the ykuD family may play an important role in cell wall biology.     

Complete primary structure of human and rabbit lactase-phlorizin hydrolase: implications for biosynthesis, membrane anchoring and evolution of the enzyme.

We report the primary structures of human and rabbit brush border membrane beta-glycosidase complexes (pre-pro-lactase-phlorizin hydrolase, or pre-pro-LPH, EC 3.2.1.23-62), as deduced from cDNA sequences. The human and rabbit primary translation products contain 1927 and 1926 amino acids respectively. Based on the data, as well as on peptide sequences and further biochemical data, we conclude that the proteins comprise five domains: (i) a cleaved signal sequence of 19 amino acids; (ii) a large 'pro' portion of 847 amino acids (rabbit), none of which appears in mature, membrane-bound LPH; (iii) the mature LPH, which contains both the lactase and phlorizin hydrolase activities in a single polypeptide chain; (iv) a membrane-spanning hydrophobic segment near the carboxy terminus, which serves as membrane anchor; and (v) a short hydrophilic segment at the carboxy terminus, which must be cytosolic (i.e. the protein has an Nout-Cin orientation). The genes have a 4-fold internal homology, suggesting that they evolved by two cycles of partial gene duplication. This repetition also implies that parts of the 'pro' portion are very similar to parts of mature LPH, and hence that the 'pro' portion may be a water-soluble beta-glycosidase with another cellular location than LPH. Our results have implications for the decline of LPH after weaning and for human adult-type alactasia, and for the evolutionary history of LPH.