Recovery of argininosuccinate lyase activity in duck delta1 crystallin

Delta-crystallin, the major soluble protein component in the avian eye lens, is homologous to argininosuccinate lyase (ASL). Two delta-crystallin isoforms exist in ducks, delta1- and delta2-crystallin, which are 94% identical in amino acid sequence. While duck delta2-crystallin (ddeltac2) has maintained ASL activity, evolution has rendered duck delta1-crystallin (ddeltac1) enzymatically inactive. Previous attempts to regenerate ASL activity in ddeltac1 by mutating the residues in the 20s (residues 22-31) and 70s (residues 74-89) loops to those found in ddeltac2 resulted in a double loop mutant (DLM) which was enzymatically inactive (Tsai, M. et al. (2004) Biochemistry 43, 11672-82). This result suggested that one or more of the remaining five amino acid substitutions in domain 1 of the DLM contributes to the loss of ASL activity in ddeltac1. In the current study, residues Met-9, Val-14, Ala-41, Ile-43, and Glu-115 were targeted for mutagenesis, either alone or in combination, to the residues found in ddeltac2. ASL activity was recovered in the DLM by changing Met-9 to Trp, and this activity is further potentiated in the DLM-M9W mutant when Glu-115 is changed to Asp. The roles of Trp-9 and Asp-115 were further investigated by site-directed mutagenesis in wild-type ddeltac2. Changing the identity of either Trp-9 or Asp-115 in ddeltac2 resulted in a dramatic drop in enzymatic activity. The loss of activity in Trp-9 mutants indicates a preference for an aromatic residue at this position. Truncation mutants of ddeltac2 in which the first 8, 9, or 14 N-terminal residues were removed displayed either decreased or no ASL activity, suggesting residues 1-14 are crucial for enzymatic activity in ddeltac2. Our kinetic studies combined with available structural data suggest that the N-terminal arm in ASL/delta2-crystallin is involved in stabilizing regions of the protein involved in substrate binding and catalysis, and in completely sequestering the substrate from the solvent.     

Mutational analysis of duck delta 2 crystallin and the structure of an inactive mutant with bound substrate provide insight into the enzymatic mechanism of argininosuccinate lyase.

The major soluble avian eye lens protein, delta crystallin, is highly homologous to the housekeeping enzyme argininosuccinate lyase (ASL). ASL is part of the urea and arginine-citrulline cycles and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. In duck lenses, there are two delta crystallin isoforms that are 94% identical in amino acid sequence. Only the delta2 isoform has maintained ASL activity and has been used to investigate the enzymatic mechanism of ASL. The role of the active site residues Ser-29, Asp-33, Asp-89, Asn-116, Thr-161, His-162, Arg-238, Thr-281, Ser-283, Asn-291, Asp-293, Glu-296, Lys-325, Asp-330, and Lys-331 have been investigated by site-directed mutagenesis, and the structure of the inactive duck delta2 crystallin (ddeltac2) mutant S283A with bound argininosuccinate was determined at 1.96 A resolution. The S283A mutation does not interfere with substrate binding, because the 280's loop (residues 270-290) is in the open conformation and Ala-283 is more than 7 A from the substrate. The substrate is bound in a different conformation to that observed previously indicating a large degree of conformational flexibility in the fumarate moiety when the 280's loop is in the open conformation. The structure of the S283A ddeltac2 mutant and mutagenesis results reveal that a complex network of interactions of both protein residues and water molecules are involved in substrate binding and specificity. Small changes even to residues not involved directly in anchoring the argininosuccinate have a significant effect on catalysis. The results suggest that either His-162 or Thr-161 are responsible for proton abstraction and reinforce the putative role of Ser-283 as the catalytic acid, although we cannot eliminate the possibility that arginine is released in an uncharged form, with the solvent providing the required proton. A detailed enzymatic mechanism of ASL/ddeltac2 is presented.     

Structural studies of duck delta 1 and delta 2 crystallin suggest conformational changes occur during catalysis

Duck delta1 and delta2 crystallin are 94% identical in amino acid sequence, and while delta2 crystallin is the duck orthologue of argininosuccinate lyase (ASL) and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, the delta1 isoform is enzymatically inactive. The crystal structures of wild type duck delta1 and delta2 crystallin have been solved at 2.2 and 2.3 A resolution, respectively, and the refinement of the turkey delta1 crystallin has been completed. These structures have been compared with two mutant duck delta2 crystallin structures. Conformational changes were observed in two regions of the N-terminal domain with intraspecies differences between the active and inactive isoforms localized to residues 23-32 and both intra- and interspecies differences localized to the loop of residues 74-89. As the residues implicated in the catalytic mechanism of delta2/ASL are all conserved in delta1, the amino acid substitutions in these two regions are hypothesized to be critical for substrate binding. A sulfate anion was found in the active site of duck delta1 crystallin. This anion, which appears to mimic the fumarate moiety of the argininosuccinate substrate, induces a rigid body movement in domain 3 and a conformational change in the loop of residues 280-290, which together would sequester the substrate from the solvent. The duck delta1 crystallin structure suggests that Ser 281, a residue strictly conserved in all members of the superfamily, could be the catalytic acid in the delta2 crystallin/ASL enzymatic mechanism.     

Expression of duck lens delta-crystallin cDNAs in yeast and bacterial hosts. Delta 2-crystallin is an active argininosuccinate lyase.

The major soluble protein in the lenses of most birds and reptiles is delta-crystallin. In chickens and ducks the delta-crystallin gene has duplicated, and in the duck both genes contribute to the protein in the lens, while in the chicken lens there is a great preponderance of the delta 1 gene product. Purified delta-crystallin has previously been shown to possess the enzymatic activity of argininosuccinate lyase. In order to determine the enzymatic properties of the two duck delta-crystallins their corresponding cDNA molecules were placed in yeast and bacterial expression plasmids. In Saccharomyces cerevisiae, the activity of each crystallin was assessed by transformation of the expression plasmids into a strain deficient for argininosuccinate lyase activity. The ability of the resulting yeast to grow on arginine deficient medium was used as a measure of enzymatic activity. Yeast expressing the duck delta 2-crystallin protein grew rapidly, while those expressing delta 1-crystallin failed to grow. Enzyme activity measurements confirmed the presence of activity in the delta 2-crystallin-expressing yeast, and no detectable activity could be demonstrated in the delta 1-crystallin-expressing yeast. Northern blotting of RNA from the transformed yeast revealed equal levels of mRNA species from the two constructs. For further analysis, the delta 2-crystallin cDNA was placed in the bacterial expression plasmid, pET-3d. The delta 2-crystallin protein produced in Escherichia coli was purified to homogeneity and analyzed to determine the kinetic properties. A Km of 0.35 mM was determined for argininosuccinate and a Vm of 3.5 mumols/min/mg was determined. These data demonstrate that, following duplication of the primordial argininosuccinate lyase gene, one of the genes maintained its role as an enzyme (delta 2-crystallin) while also serving as a crystallin and the other has evolved to specialize as a structural protein in the lens (delta 1-crystallin), presumably losing most or all of its catalytic capacity.     

Three-dimensional structure of the argininosuccinate lyase frequently complementing allele Q286R.

Argininosuccinate lyase (ASL) catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate, a reaction involved in the biosynthesis of arginine in all species and in the production of urea in ureotelic species. In humans, mutations in the enzyme result in the autosomal recessive disorder argininosuccinic aciduria. Intragenic complementation has been demonstrated to occur at the ASL locus, with two distinct classes of ASL-deficient strains having been identified, the frequent and high-activity complementers. The frequent complementers participate in the majority of the complementation events observed and were found to be either homozygous or heterozygous for a glutamine to arginine mutation at residue 286. The three-dimensional structure of the frequently complementing allele Q286R has been determined at 2.65 A resolution. This is the first high-resolution structure of human ASL. Comparison of this structure with the structures of wild-type and mutant duck delta1 and delta2 crystallins suggests that the Q286R mutation may sterically and/or electrostatically hinder a conformational change in the 280's loop (residues 270-290) and domain 3 that is thought to be necessary for catalysis to occur. The comparison also suggests that residues other than R33, F333, and D337 play a role in maintaining the structural integrity of domain 1 and reinforces the suggestion that residues 74-89 require a particular conformation for catalysis. The electron density has enabled the structure of residues 6-18 to be modeled for the first time. Residues 7-9 and 15-18 are in type IV beta-turns and are connected by a loop. The conformation observed is stabilized, in part, by a salt bridge between the side chains of R12 and D18. Although the disease causing mutation R12Q would disrupt this salt bridge, it is unclear why this mutation has such a significant effect on the catalytic activity as residues 1-18 are disordered in all other delta-crystallin structures determined to date.     

Sequence analysis of pigeon delta-crystallin gene and its deduced primary structure. Comparison of avian delta-crystallins with and without endogenous argininosuccinate lyase activity.

delta-Crystallin is a major lens protein present in the avian and reptilian lenses. To facilitate the cloning of the delta-crystallin gene, cDNA was constructed from the poly(A)+ RNA of pigeon lenses, amplified by the polymerase chain reaction (PCR). The PCR product was then subcloned into pUC19 vector and transformed into E. coli strain JM109. Plasmids purified from the positive clones were prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method. Sequencing two clones, containing 1.4 kb DNA inserts coding for delta-crystallin allowed the construction of a complete, full-length reading frame of 1,417 bp covering a deduced protein sequence of 466 amino acids, including the universal translation-initiating methionine. The pigeon delta-crystallin shows 88, 83 and 69% sequence identity to duck delta 2, chicken delta 1 crystallins and human argininosuccinate lyase respectively. It is also shown that, in contrast to duck delta 2 crystallin which has a high argininosuccinate lyase activity, pigeon delta-crystallin appears to contain very low activity of this enzyme, despite the fact that they share a highly homologous structure. A structural comparison of delta-crystallins with or without enzymatic activity suggested several amino acid replacements which may account for the loss of argininosuccinate lyase activity in the lenses of certain avian species.     

Domain exchange experiments in duck delta-crystallins: functional and evolutionary implications

Delta-crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two delta crystallins, denoted delta1 and delta2. Duck delta2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to delta2/ASL in the amino acid sequence, delta1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras--221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, delta1 or delta2, respectively--were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to delta1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in delta1. The thermodynamic characterization of human ASL (hASL) and duck delta1 and delta2 indicate that delta crystallins are slightly less stable than hASL, with the delta1 being the least stable. The deltaGs of unfolding are 57.25, 63.13, and 70.71 kcal mol(-1) for delta1, delta2, and hASL, respectively. This result was unexpected, and we speculate that delta crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein-protein and protein-solvent interactions.     

Mutational analysis of amino acid residues involved in argininosuccinate lyase activity in duck delta II crystallin

Delta-crystallins are the major structural eye lens proteins of most birds and reptiles and are direct homologues of the urea cycle enzyme argininosuccinate lyase. There are two isoforms of delta-crystallin, delta Iota and delta IotaIota, but only delta IotaIota crystallin exhibits argininosuccinate lyase (ASL) activity. At the onset of this study, the structure of argininosuccinate lyase/delta IotaIota crystallin with bound inhibitor or substrate analogue was not available. Biochemical and X-ray crystallographic studies had suggested that H162 may function as the catalytic base in the argininosuccinate lyase/delta IotaIota crystallin reaction mechanism, either directly or indirectly through the activation of a water molecule. The identity of the catalytic acid was unknown. In this study, the argininosuccinate substrate was modeled into the active site of duck delta IotaIota crystallin, using the coordinates of an inhibitor-bound Escherichia coli fumarase C structure to orient the fumarate moiety of the substrate. The model served as a means of identifying active site residues which are positioned to potentially participate in substrate binding and/or catalysis. On the basis of the results of the modeling, site-directed mutagenesis was performed on several amino acids, and the kinetic and thermodynamic properties of each mutant were determined. Kinetic studies reveal that five residues, R115, N116, T161, S283, and E296, are essential for catalytic activity. Determination of the free energy of unfolding/refolding of wild-type and mutant delta II crystallins revealed that all constructs exhibit similar thermodynamic stabilities. During the course of this work, the structure of an inactive delta IotaIota crystallin mutant with bound substrate was solved [Vallee et al. (1999) Biochemistry 38, 2425-2434], which has allowed the kinetic data to be interpreted on a structural basis.     

Gene conversion and splice-site slippage in the argininosuccinate lyases/delta-crystallins of the duck lens: members of an enzyme superfamily

Argininosuccinate lyase(ASL)/delta-crystallin is a prominent example of an enzyme-crystallin with roles as both a catalyst and a major structural component of the eye lens in birds and reptiles. In chicken it appears that gene duplication and separation of function may have occurred with one gene product acting primarily as a crystallin and one primarily as an enzyme. However, two delta-crystallin-encoding genes are abundantly expressed in the lens of the embryonic duck (Anas platyrhynchos) which has extremely high ASL activity. Here the isolation and sequence analysis of full length cDNA clones for both duck delta-crystallins are described. The two delta-crystallins are highly similar (94% identical in predicted aa sequence), probably as a result of gene conversion. However, the cDNA for duck delta 2-crystallin contains an in-frame insertion of two codons, probably the result of a recent intron boundary slippage. ASL/delta-crystallin belongs to a superfamily of lyases, including fumarases, aspartases and adenylosuccinate lyase which possess some highly conserved blocks of aa sequence. There may be some clues to the tertiary structures of these conserved motifs in otherwise unrelated proteins for which three-dimensional structures are known.     

Crystal structure of an inactive duck delta II crystallin mutant with bound argininosuccinate

Delta-crystallin, the major soluble protein component of avian and reptilian eye lenses, is highly homologous to the urea cycle enzyme, argininosuccinate lyase (ASL). In duck lenses, there are two highly homologous delta crystallins, delta I and delta II, that are 94% identical in amino acid sequence. While delta II crystallin has been shown to exhibit ASL activity in vitro, delta I is enzymatically inactive. The X-ray structure of a His to Asn mutant of duck delta II crystallin (H162N) with bound argininosuccinate has been determined to 2.3 A resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft formed by three different monomers in the tetramer. The active site of the H162N mutant structure reveals that the side chain of Glu 296 has a different orientation relative to the homologous residue in the H91N mutant structure [Abu-Abed et al. (1997) Biochemistry 36, 14012-14022]. This shift results in the loss of the hydrogen bond between His 162 and Glu 296 seen in the H91N and turkey delta I crystallin structures; this H-bond is believed to be crucial for the catalytic mechanism of ASL/delta II crystallin. Argininosuccinate was found to be bound to residues in each of the three monomers that form the active site. The fumarate moiety is oriented toward active site residues His 162 and Glu 296 and other residues that are part of two of the three highly conserved regions of amino acid sequence in the superfamily, while the arginine moiety of the substrate is oriented toward residues which belong to either domain 1 or domain 2. The analysis of the structure reveals that significant conformational changes occur on substrate binding. The comparison of this structure with the inactive turkey delta I crystallin reveals that the conformation of domain 1 is crucial for substrate affinity and that the delta I protein is almost certainly inactive because it can no longer bind the substrate.     

Distinct interactions of αA-crystallin with homologous substrate proteins, δ-crystallin and argininosuccinate lyase, under thermal stress

δ-Crystallin is a taxon-specific eye lens protein that was recruited from argininosuccinate lyase (ASL) through gene sharing. ASL is a metabolic enzyme that catalyzes the reversible conversion of argininosuccinate into arginine and fumarate and shares about 70% sequence identity and similar overall topology with δ-crystallin. ASL has a lower thermal stability than δ-crystallin. In this study, we show that the small heat shock protein, αA-crystallin, functions as a molecular chaperone, and enhanced thermal stability of both δ-crystallin and ASL. The stoichiometry for efficient protection of the two substrate proteins by αA-crystallin was determined by slowly increasing the temperature. N- or C-terminal truncated mutants of δ-crystallin co-incubated with αA-crystallin showed higher thermal stability than wild-type enzyme, and the stoichiometry for efficient protection was the same. Thermal unfolding of δ-crystallin or ASL in the presence of αA-crystallin followed a similar three-state model, as determined by circular dichroism analyses. A stable intermediate which retained about 30% α-helical structure was observed. Protection from thermal denaturation by αA-crystallin was by interaction with partly unfolded ASL or δ-crystallin to form high molecular weight heteroligomers, as judged by size-exclusive chromatography and SDS-PAGE analyses. Aggregate formation of ASL was significantly reduced in the presence of αA-crystallin. The extent of protection of ASL and δ-crystallin at different ratios of αA-crystallin were described by hyperbolic and sigmoidal curves, respectively. These results suggest the preferential recognition of partly unfolded ASL by αA-crystallin. In contrast, unstable δ-crystallin might trigger a cooperative interaction by higher stoichiometries of αA-crystallin leading to fuller protection. The different interactions of αA-crystallin with the two homologous but functionally different substrate proteins show its behavior as a chaperone is variable.     

Two delta-crystallin polypeptides are derived from a cloned delta 1-crystallin cDNA

Previous studies have shown that there are 2 similar delta-crystallin genes (delta 1 and delta 2) and at least 2 delta-crystallin polypeptides in the chicken eye lens. We show here that both delta-crystallin polypeptides can be synthesized from mRNA transcribed in vitro from a cloned delta 1-crystallin cDNA. Both polypeptides co-migrate in SDS-urea-polyacrylamide electrophoresis with their authentic counterparts isolated from 15-day-old embryonic chicken lenses, and both react with sheep anti-chicken delta-crystallin serum. Screening nearly 900 delta-crystallin cDNA clones from a 15-day-old embryonic lens library with an oligonucleotide probe specific for exon 2 of the delta 2-crystallin gene failed to detect any delta 2 cDNA clones, indicating that the delta 2 gene produces little or no mRNA in the lens at this stage of development. Our results suggest that both of the observed delta-crystallin polypeptides are derived from mRNA transcribed from the delta 1 gene, with heterogeneity arising at the translational or co-translational level.     

α-Crystallin protects human arginosuccinate lyase activity under freeze-thaw conditions

Argininosuccinate lyase (ASL) catalyzes the conversion of argininosuccinate into arginine and fumarate, a key step in the biosynthesis of urea and arginine. ASL is a tetrameric enzyme but it dissociates into inactive dimers under low temperature conditions. This study investigates the inactivation process under low temperature conditions. Inactivation was caused by dissociation of tetrameric ASL into dimers, with increased exposure of hydrophobic areas without disturbance of the secondary structure or the microenvironment surrounding the key tryptophan residues. Most activity was retained when temperatures were changed at a rate of >1 °C/min, whilst freezing or thawing more slowly resulted in greater loss of activity. Inactivation was reduced by inclusion of α-crystallin, a structural protein found in ocular lenses and a member of the small heat-shock protein family, by stabilization of the ASL quaternary structure. In addition, α-crystallin was able to restore the function of ASL that had been inactivated by slow freezing and thawing. The effect of α-crystallin was similar to that of bovine serum albumin, suggesting that both proteins exerted their effects by hydrophobic interactions. α-Crystallin therefore acts as a cryo-preservative that protects ASL activity during freezing and thawing.     

Biochemical characterization of crystallins from pigeon lenses: structural and sequence analysis of pigeon delta-crystallin.

Crystallins from pigeon eye lenses were isolated and purified by gel-permeation chromatography and characterized by gel electrophoresis, amino-acid composition and sequence analysis. Alpha- and beta-crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta-crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of the gel filtration column. Pigeon delta-crystallin, similar to duck and reptilian delta-crystallins, exists as a tetrameric structure of about 200 kDa in the native form and is composed of one major subunit of 50 kDa with heterogeneous isoelectric points spreading in a range of 4.7 to 6.8. In contrast to those obtained from duck, goose and caiman, delta-crystallin isolated from the pigeon lens possessed very little argininosuccinate lyase activity. However, pigeon delta-crystallin can still cross-react with the antibody against enzymically active duck delta-crystallin as revealed by the sensitive immunoblotting technique. It was also shown that the delta-crystallin content of the total pigeon soluble proteins decreased with the age of the animal. Structural analysis of purified delta-crystallin fraction was made with respect to its amino-acid composition and protein primary sequence. N-terminal sequence analysis indicated the presence of blocked amino-termini in all crystallin fractions of pigeon lenses. Therefore, a sequence analysis of PCR (polymerase chain reaction) amplified delta-crystallin cDNA was employed to deduce the protein sequence of this crystallin. Structural comparison of delta-crystallin sequences from pigeon, chicken and duck lenses casts some doubts on the recent claim that His-89-->Gln mutation in the chicken delta-crystallin may account for the loss of argininosuccinate lyase activity in this avian species, as compared to high enzymic activity in the duck crystallin (Barbosa et al. (1991) J. Biol. Chem. 266, 5286-5290).