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The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al. (1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.

The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed "hemicellulose." One such hemicellulose is xyloglucan, which displays a ␤-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligandfree and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (␤/␣) 8 and ␤-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the ␤-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (␤/␣) 8 GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.

Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

Protein engineering on trypanosomal triosephosphate isomerase (TIM) converted this oligomeric enzyme into a stable, monomeric protein that is enzymatically active.Wild-type TIM consists of two identical subunits that form a very tight dimer involving interactions of 32 residues of each subunit. By replacing 15 residues of the major interface loop by another 8-residue fragment, a variant was constructed that is a stable and monomeric protein with TIM activity. The length, sequence, and conformation of the designed fragment were suggested by extensive modeling.The (,B/a)g protein fold, commonly known as the triosephosphate isomerase (TIM) fold, seems to be nature's favorite choice of a scaffold for enzymatic activities as %20 different enzymes have, so far, been shown to have this fold (1, 2). The 3-strands form an internal 1-barrel consisting of 8 parallel ,3-strands (strands 1-8), covered on the outside by 8 a-helices (helices [1][2][3][4][5][6][7][8]. The connections between 3-strands and the subsequent a-helices are referred to as loops 1-8. The side chains of some of these loop residues form the active site of the TIM barrel enzymes and novel active sites can therefore theoretically be constructed at the C terminus of the ,/strands without altering the overall fold. Our goal is to design activities on this (,3/a)8 framework. Interestingly, most of the known (/3/a)8 proteins are part of homo-or heterooligomers. For our studies, TIM from Trypanosoma brucei was chosen as the model protein because the crystal structure is known at 1.83 A resolution (3). TIM forms very stable dimers with high catalytic activity. These properties have hindered the accurate measurements of the activity of individual monomers; the monomers have been postulated to be inactive in both renaturation (4, 5) and mutagenesis (6) studies, whereas matrix-bound monomers have been reported to retain activity (7). No allosteric control or cooperativity has been found between the two subunits (8, 9). The Kd of the TIM dimer is not known, but based on the low protein concentration in the activity assay, and assuming that the monomer is much less active than the dimer, then the Kd value can be estimated to be <10 pM. The first step in our protein engineering scheme has been to convert the dimeric TIM to a monomeric protein to (i) facilitate the subsequent design of active sites, (ii) examine the functional and structural importance of dimer formation, and (iii) have the principal possibility to obtain an active monomer. Only few such mutagenesis experiments of other proteins have been described. One example is the protein hormone insulin, in which it has been shown that single and double amino acid alterations of interface residues led to considerable variations in the association of the insulin monomers (10). The dimeric ACro DNA binding protein has been converted to a stable monomer that binds to DNA (11). Both cases involve small nonenzyme proteins on the order of 60 residues and insulin is, in fact, functional as a monomer. Single amino...

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.