Aldolase crystallization

Muscle aldolase crystallizes as a simple protein with a molecular weight near 150,000. The corresponding enzyme from yeast (molecular weight, 122,000) has recently been crystallized as an inactive mercury salt, which requires removal of the mercury and addition of divalent zinc, iron, or cobalt for activation. Similar metal requirements have been found for aldolases from other microorganisms. 

Antibodies produced by this procedure were screened for their ability to react with the hapten to form the vinylogous amide 6, which has a convenient UV chromophore near 318nm, clear of the main protein absorption. Two antibodies selected in this way catalysed the expected aldol reaction of acetone with aldehyde 7 by way of the enamine 8 (Scheme 3) the remainder did not. These two effective aldolase mimics have been studied in some detail, and a crystal structure is available for (a Fab fragment of) one of them.126,281... 

Folate biosynthesis has also been studied in plants and the dihydroneopterin aldolase from Arabidopsis thaliana has been crystallized and its structure determined the construction of the active site has similarities with those of other... 

II aldolases FucA and RhuA from E. coli have been crystallized solution of their spatial structures confirmed a close similarity in their overall fold [14]. Both enzymes are homotetramers in which subunits are arranged in C4 symmetry. The active site is assembled in deep clefts at the interface between adjacent subunits, and the catalytic zinc ion is tightly coordinated by three His residues. From X-ray... 

The L-rhamnulose 1-phosphate aldolase (RhuA EC 4.1.2.19) is found in the microbial degradation of L-rhamnose which, after conversion into the corresponding ketose 1-phosphate 44, is cleaved into 41 and L-lactaldehyde (l-16). The RhuA has been isolated from E. coli [336-339], and characterized as a metallo-protein [194,340,341]. Cloning was reported for the E. coli [342,343] and Salmonella typhimurium [344] genes, and construction of an efficient overexpression system [195,220] has set the stage for crystallization of the homotetrameric E. coli protein for the purposes of an X-ray structure analysis [345]. 

Functionally and mechanistically reminiscent of the pyruvate lyases, the 2-deoxy-D-ribose 5-phosphate (121) aldolase (RibA EC 4.1.2.4) [363] is involved in the deoxynucleotide metabolism where it catalyzes the addition of acetaldehyde (122) to D-glyceraldehyde 3-phosphate (12) via the transient formation of a lysine Schiff base intermediate (class I). Hence, it is a unique aldolase in that it uses two aldehydic substrates both as the aldol donor and acceptor components. RibA enzymes from several microbial and animal sources have been purified [363-365], and those from Lactobacillus plantarum and E. coli could be induced to crystallization [365-367]. In addition, the E. coli RibA has been cloned [368] and overexpressed. It has a usefully high specific activity [369] of 58 Umg-1 and high affinity for acetaldehyde as the natural aldol donor component (Km = 1.7 mM) [370]. The equilibrium constant for the formation of 121 of 2 x 10M does not strongly favor synthesis. Interestingly, the enzyme s relaxed acceptor specificity allows for substitution of both cosubstrates propional-dehyde 111, acetone 123, or fluoroacetone 124 can replace 122 as the donor [370,371], and a number of aldehydes up to a chain length of 4 non-hydrogen atoms are tolerated as the acceptor moiety (Table 6). 

Fermentation methods for synthesis and resolution Reaction with cyclic lactam intermediates Reaction with glycine and aldolase Fractional crystallization... 

The new aldolase differs from all other existing ones with respect to the location of its active site in relation to its secondary structure and still displays enantiofacial discrimination during aldol addition. Modification of substrate specificity is achieved by altering the position of the active site lysine from one /3-strand to a neighboring strand rather than by modification of the substrate recognition site. Determination of the 3D crystal structure of the wild type and the double mutant demonstrated how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate. It is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. 

G. Schneider, Crystal structure of transaldolase B from Escherichia coli suggests a circular permutation of the a/ P-barrel within the class I aldolase family, Structure 1996, 4, 715-724. 

Cooper, S. J., Leonard, G. A., McSweeney, S. M., Thompson, A. W., Naismith, J. H., Qamar, S., et al. The Crystal Structure of a Class II Fructose-1,6-Bisphosphate Aldolase Shows a Novel Binuclear Metal-Binding Active Site Embedded in a Familiar Fold. Structure 1996, 4, 1303-1315. 

Hall, D. R., Leonard, G. A., Reed, C. D., Watt, C. I., Berry, A., Hunter, W. N. The Crystal Structure of Escherichia coli Class II Fructose-1,6-Bisphosphate Aldolase in Complex with Phosphoglycolohydroxamate Reveals Details of Mechanism and Specificity. J. Mol. Biol. 1999, 287, 383-394. 

SB Sobolov, A Bartoszko-Malik, TR Oeschger, MM Montelbano. Cross-linked enzyme crystals of fructose diphosphate aldolase development as a biocatalyst for synthesis. Tetrahedron Lett 35 7751-7754, 1994. 

Like transketolase, transaldolase (TA, E.C. 2.2.1.2) is an enzyme in the oxidative pentose phosphate pathway. TA is a class one lyase that operates through a Schiff-base intermediate and catalyzes the transfer of the C(l)-C(3) aldol unit from D-sedoheptulose 7-phosphate to glyceraldehyde-3-phosphate (G3P) to produce D-Fru 6-P and D-erythrose 4-phosphate (Scheme 5.59). TA from human as well as microbial sources have been cloned.110 111 The crystal structure of the E. coliu and human112 transaldolases have been reported and its similarity to the aldolases is apparent, since it consists of an eight-stranded (o /(3)s or TIM barrel domain as is common to the aldolases. As well, the active site lysine residue that forms a Schiff base with the substrate was identified.14112 Thus, both structurally and mechanistically it is related to the type I class of aldolases. 

Machajewski TD, Wong CH. The catalytic asymmetric aldol reaction. Angew. Chem. Int. Ed. Engl. 2000 39(8) 1352—1375. Heine A, Luz JG, Wong CH, Wilson lA. Analysis of the class 1 aldolase binding site architecture based on tbe crystal stracture of 2-deoxyribose-5-phosphate aldolase at 0.99A resolution. J. Mol. Biol. 2004 343(4) 1019-1034. 

S. J. Cooper, G.A. Leonard, S.M. McSweeney, A.W. Thompson, J.H. Naismith, S. Qamar, A. Plater, A. Berry, and W.N. Hunter. 1996. The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel hinuclear metalbinding active site embedded in a familiar fold Structure 4 1303-1315. (PubMed)... 

Lorentzen, E., E. Pohl, R Zwart, A. Stark, R. B. Russell, T. Knura, R. Hensel, and B. Siebers. 2003. Crystal structure of an archaeal class I aldolase and the evolution of (betaalpha) 8 barrel proteins. J Biol Chem 278 47253-60. 

---