Engineered amadoriase II exhibiting expanded substrate range

Amadori compounds are ubiquitous in vivo as well as in food and have been implicated in diabetic complications and aging. In recent years, fructosyl amine oxidases (FAOXs) which cleave Amadori products are gaining increasing attention. Until now, however, all FAOXs can only react with small glycated substrates (such as fructosyl amino acids or dipeptides), which has hindered the applications of this new class of enzymes in diagnosis, therapeutics, and detergents. In this study, Aspergillus fumigatus amadoriase II was engineered with the aim to expand its substrate range, using a heat-inducible autolytic vector and fructosyl–polylysine (3–13 lysines) as an intermediate-sized model substrate. After two rounds of directed evolution, a mutant (SII-82) was obtained that showed an 8.78-fold increase in the activity toward fructosyl–polylysine and which also performed several fold better than the wild-type on real gravy stains at concentrations of 10–100 μg/ml (parts per million). Mutational analyses revealed useful clues for altering the substrate-binding pocket. This study suggests that it is possible to manipulate fructosyl amine oxidases to accommodate larger substrates, and that mutant SII-82 might serve as a template for further engineering.     

Crystal structure of the deglycating enzyme fructosamine oxidase (amadoriase II)

Fructosamine oxidases (FAOX) catalyze the oxidative deglycation of low molecular weight fructosamines (Amadori products). These proteins are of interest in developing an enzyme to deglycate proteins implicated in diabetic complications. We report here the crystal structures of FAOX-II from the fungi Aspergillus fumigatus, in free form and in complex with the inhibitor fructosyl-thioacetate, at 1.75 and 1.6A resolution, respectively. FAOX-II is a two domain FAD-enzyme with an overall topology that is most similar to that of monomeric sarcosine oxidase. Active site residues Tyr-60, Arg-112 and Lys-368 bind the carboxylic portion of the fructosamine, whereas Glu-280 and Arg-411 bind the fructosyl portion. From structure-guided sequence comparison, Glu-280 was identified as a signature residue for FAOX activity. Two flexible surface loops become ordered upon binding of the inhibitor in a catalytic site that is about 12A deep, providing an explanation for the very low activity of FAOX enzymes toward protein-bound fructosamines, which would have difficulty accessing the active site. Structure-based mutagenesis showed that substitution of Glu-280 and Arg-411 eliminates enzyme activity. In contrast, modification of other active site residues or of amino acids in the flexible active site loops has little effect, highlighting these regions as potential targets in designing an enzyme that will accept larger substrates.     

Rational protein crystallization by mutational surface engineering

Protein crystallization constitutes a limiting step in structure determination by X-ray diffraction. Even if single crystals are available, inadequate physical quality may seriously limit the resolution of the available data and consequently the accuracy of the atomic model. Recent studies show that targeted mutagenesis of surface patches containing residues with large flexible side chains and their replacement with smaller amino acids lead to effective preparation of X-ray quality crystals of proteins otherwise recalcitrant to crystallization. Furthermore, this technique can also be used to obtain crystals of superior quality as compared to those grown for the wild-type protein, sometimes increasing the effective resolution by as much as 1 A or more. Several recent examples of this new methodology suggest that the method has the potential to become a routine tool in protein crystallography.     

Temperature-dependent negative cooperativity among angiotensin II receptors of isolated rat glomeruli

The binding characteristics of angiotensin II to isolated rat glomeruli were studied at various temperatures from 15 to 37 degrees C using 125I-labeled angiotensin II. The remarkable features of the binding at 37 degrees C, compared with those at lower temperatures, were (1) decreased maximal binding due to increased dissociation rate, (2) short duration of the steady state, which was, however, sufficient for performing steady-state binding assays, and (3) marked curvilinear Scatchard plots with upward concavity. The difference between the dissociation rates caused by dilution only and by dilution plus cold angiotensin II increased with increase in temperature. From these results, we conclude that the site-site interactions of a negatively cooperative type, which are negligible at lower temperatures, exist among rat glomerular angiotensin II receptors at the physiological temperature, and that the binding study may as well be performed at 37 degrees C for the purpose of investigating quantitative correlation between the hormone binding and its biological effect.     

Alteration of substrate selectivity through mutation of two arginine residues in the binding site of amadoriase II from Aspergillus sp

Amadoriases I and II are deglycation isoenzymes from Aspergillus sp. of potential relevance for treatment of diabetic complications resulting from excessive protein glycation. Amadoriase II has a preference for anionic substrate with a K(m) of 0.23 and 2.53 mM for fructosylglycine and fructosylpropylamine, respectively. In contrast, the corresponding K(m) values for amadoriase I are 9.75 and 0.023 mM, respectively. Chemical modification of amadoriase II with p-hydroxyphenylglyoxal, a specific arginine-modifying reagent, resulted in an inhibition of enzyme activity toward fructosylglycine, while having less effect on the enzymatic activity toward fructosylpropylamine. Peptide mapping and subsequent mass spectrometry analysis suggest that Arg(112) is one of the sites of p-hydroxyphenylglyoxal modification. Sequence alignment between amadoriase I and amadoriase II revealed that two glutamic acids in amadoriase I align to Arg(112) and Arg(114) in amadoriase II. Site-directed mutation of amadoriase II (R112E, R114E) resulted in reversal of the enzymatic activities toward fructosylglycine and fructosylpropylamine. Our results suggested that Arg(112) and Arg(114) are responsible for the high affinity of amadoriase II toward anionic substrates and determine the substrate selectivity of the enzyme.     

Plant protoplasts and genetic engineering II

Book Review

Plant protoplasts and genetic engineering IIY.P.S. Bajaj (Ed.), (Biotechnology in Agriculture and Forestry, Vol. 9). Berlin: Springer-Verlag, 1989. 510 pages. DM398.00. ISBN 3-540-50789-2     

Structural and mutational studies of a hyperthermophilic intein from DNA polymerase II of Pyrococcus abyssi

Protein splicing is a precise self-catalyzed process in which an intein excises itself from a precursor with the concomitant ligation of the flanking polypeptides (exteins). Protein splicing proceeds through a four-step reaction but the catalytic mechanism is not fully understood at the atomic level. We report the solution NMR structures of the hyperthermophilic Pyrococcus abyssi PolII intein, which has a noncanonical C-terminal glutamine instead of an asparagine. The NMR structures were determined to a backbone root mean square deviation of 0.46 ? and a heavy atom root mean square deviation of 0.93 ?. The Pab PolII intein has a common HINT (hedgehog intein) fold but contains an extra β-hairpin that is unique in the structures of thermophilic inteins. The NMR structures also show that the Pab PolII intein has a long and disordered loop in place of an endonuclease domain. The N-terminal Cys-1 amide is hydrogen bonded to the Thr-90 hydroxyl in the conserved block-B TXXH motif and the Cys-1 thiol forms a hydrogen bond with the block F Ser-166. Mutating Thr-90 to Ala dramatically slows N-terminal cleavage, supporting its pivotal role in promoting the N-S acyl shift. Mutagenesis also showed that Thr-90 and His-93 are synergistic in catalyzing the N-S acyl shift. The block F Ser-166 plays an important role in coordinating the steps of protein splicing. NMR spin relaxation indicates that the Pab PolII intein is significantly more rigid than mesophilic inteins, which may contribute to the higher optimal temperature for protein splicing.     

Dynamics and cooperativity of Trp-cage folding

In this study, multiple independent molecular dynamics (MD) simulations on Trp-cage folding were performed at 300, 325 and 375 K using generalized Born (GB) implicit solvent model. The orientational movement of the side-chain of Trp6 to form a hydrophobic core with 3₁₀-helix was observed. The breaking/formation of a salt bridge between Asp9 and Arg16 was proposed to be the prerequisite for Trp-cage folding/refolding. Our results demonstrate that the cooperation between the salt bridge and the Trp6 orientation leads to a stable tertiary structure of Trp-cage. Analyses on backbone concerted motions at different temperatures indicate that interactions between Trp6 and 3₁₀-helix & Pro18 and between Pro12 and Pro17 & Pro18 are weakened at 375 K but strengthened at lower temperatures, suggesting that they could be the potential driving force of hydrophobic collapse.     

Computer-based learning of cooperativity and allostery

The aim of this article is to facilitate the understanding ofenzyme cooperativity and allostery by undergraduate and postgraduatestudents with the aid of a graphic microcomputer. For this purposethe molecular models of Monod-Wyman-Changeux (MWC) and of Koshland-Nemethy-Filmer(KNF) are tested by showing how the different plots, direct,reciprocal, Scatchard and Hill, vary as do the parameters consideredin these models. The programs used (one for each model) progressfrom easy aspects to complicated ones without the interventionof the user (student). Nevertheless, ultimately, with the MWCmodel, after the introduction of heterotropic effectors, theusers can select the parameters in order to further their knowledge.This can be useful also for testing the kinetic behaviour ofmultisubunit enzymes which present cooperativity and which havebeen extensively described in the literature. Received on March 28, 1985; accepted on June 20, 1985     

Ultraviolet mutagenesis in bacteriophage T4. II. Photoreversal of mutational lesions

Drake, John W. (University of Illinois, Urbana). Ultraviolet mutagenesis in bacteriophage T4. II. Photoreversal of mutational lesions. J. Bacteriol. 92:144-147. 1966.-T4r mutations were induced by ultraviolet irradiation of extracellular phage particles, using a phage mutant, v, which is particularly susceptible to photoreactivation. Most of the induced r mutations could be subsequently photoreversed intracellularly with white light. Ultraviolet irradiation induces both transitions and sign mutations, and both were susceptible to photoreversal. The results suggest that two very different types of mutational lesions may arise from a common type of photochemical lesion.     

Loop II of DNA polymerase beta is important for polymerization activity and fidelity

The accurate replication and transmission of genetic information is critical in the life of an organism. During its entire lifespan, the genetic information is constantly under attack from endogenous and exogenous sources of damage. To ensure that the content of its genetic information is faithfully preserved for synthesis and transmission, eukaryotic cells have developed a complex system of genomic quality control. Key players in this process are DNA polymerases, the enzymes responsible for synthesizing the DNA, because errors introduced into the genome by polymerase can result in mutations. We use DNA polymerase beta (pol β) as a model system to investigate mechanisms of preserving fidelity during nucleotide incorporation. In the study described here, we characterized the role that loop II of pol β plays in maintaining the activity and fidelity of pol β. We report here that the absence or shortening of loop II compromises the catalytic activity of pol β. Our data also show that loop variants of a specific length have a lower fidelity when compared to the wild-type polymerase. Taken together, our results indicate that loop II is important for the catalytic activity and fidelity of pol β.     

Energetics and cooperativity of the hydrogen bonding and anchor interactions that bind peptides to MHC class II protein

The complexity of the interaction between major histocompatibility complex class II (MHC II) proteins and peptide ligands has been revealed through structural studies and crystallographic characterization. Peptides bind through side-chain "anchor" interactions with MHC II pockets and an extensive array of genetically conserved hydrogen bonds to the peptide backbone. Here we quantitatively investigate the kinetic hierarchy of these interactions. We present results detailing the impact of single side-chain mutations of peptide anchor residues on dissociation rates, utilizing two I-A(d)-restricted peptides, one of which has a known crystal structure, and 24 natural and non-natural amino acid mutant variants of these peptides. We find that the N-terminal P1, P4 and P6 anchor-pocket interactions can make significant contributions to binding stability. We also investigate the interactions of these peptides with four I-A(d) MHC II proteins, each mutated to disrupt conserved hydrogen bonds to the peptide backbone. These complexes exhibit kinetic behavior suggesting that binding energy is disproportionately invested near the peptide N terminus for backbone hydrogen bonds. We then evaluate the effects of simultaneously modifying both anchor and hydrogen bonding interactions. A quantitative analysis of 71 double mutant cycles reveals that there is little apparent cooperativity between anchor residue interactions and hydrogen bonds, even when they are directly adjacent (<5A).     

Conformation and cooperativity in hemoglobin.

19-F and 31-P nuclear magnetic resonance (NMR) spectroscopy have been used to study the ligand binding process in human hemoglobin. 19-F nuclear magnetic resonance studies of hemoglobin specifically trifluoroacetonylated at cysteine-beta93 have permitted observation and characterization of molecular species containing two and three ligands. The behavior of these intermediate species in response to changes in pH and organic phosphate concentration is not completely consistent with any of the current theories of allostery. A model consistent with the 19-F and 31-P NMR data is proposed.