Biomedical subjects

Loredano Pollegioni

Publications and source records attributed to Loredano Pollegioni.

At least 19 recordsLinked to original sources

PubMed2006

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity.

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.

Amino Acid Substitution↗

PubMed2006

Structural and kinetic analyses of the H121A mutant of cholesterol oxidase.

Cholesterol oxidase is a monomeric flavoenzyme that catalyses the oxidation of cholesterol to cholest-5-en-3-one followed by isomerization to cholest-4-en-3-one. The enzyme from Brevibacterium sterolicum contains the FAD cofactor covalently bound to His121. It was previously demonstrated that the H121A substitution results in a approximately 100 mV decrease in the midpoint redox potential and a approximately 40-fold decrease in turnover number compared to wild-type enzyme [Motteran, Pilone, Molla, Ghisla and Pollegioni (2001) Journal of Biological Chemistry 276, 18024-18030]. A detailed kinetic analysis of the H121A mutant enzyme shows that the decrease in turnover number is largely due to a corresponding decrease in the rate constant of flavin reduction, whilst the re-oxidation reaction is only marginally altered and the isomerization reaction is not affected by the substitution and precedes product dissociation. The X-ray structure of the mutant protein, determined to 1.7 A resolution (1 A identical with 0.1 nm), reveals only minor changes in the overall fold of the protein, namely: two loops have slight movements and a tryptophan residue changes conformation by a rotation of 180 degrees about chi1 compared to the native enzyme. Comparison of the isoalloxazine ring moiety of the FAD cofactor between the structures of the native and mutant proteins shows a change from a non-planar to a planar geometry (resulting in a more tetrahedral-like geometry for N5). This change is proposed to be a major factor contributing to the observed alteration in redox potential. Since a similar distortion of the flavin has not been observed in other covalent flavoproteins, it is proposed to represent a specific mode to facilitate flavin reduction in covalent cholesterol oxidase.

Amino Acid Substitution↗

PubMed2006

Glia-derived D-serine controls NMDA receptor activity and synaptic memory.

The NMDA receptor is a key player in excitatory transmission and synaptic plasticity in the central nervous system. Its activation requires the binding of both glutamate and a co-agonist like D-serine to its glycine site. As D-serine is released exclusively by astrocytes, we studied the physiological impact of the glial environment on NMDA receptor-dependent activity and plasticity. To this end, we took advantage of the changing astrocytic ensheathing of neurons occurring in the supraoptic nucleus during lactation. We provide direct evidence that in this hypothalamic structure the endogenous co-agonist of NMDA receptors is D-serine and not glycine. Consequently, the degree of astrocytic coverage of neurons governs the level of glycine site occupancy on the NMDA receptor, thereby affecting their availability for activation and thus the activity dependence of long-term synaptic changes. Such a contribution of astrocytes to synaptic metaplasticity fuels the emerging concept that astrocytes are dynamic partners of brain signaling.

Animals↗

PubMed2006

Tryptophan 243 affects interprotein contacts, cofactor binding and stability in D-amino acid oxidase from Rhodotorula gracilis.

The flavoenzyme d-amino acid oxidase from Rhodotorula gracilis is a homodimeric protein whose dimeric state has been proposed to occur as a result of (a) the electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3'' of the other monomer, and (b) the interaction of residues (e.g. Trp243) belonging to the two monomers at the mixed interface region. The role of Trp243 was investigated by substituting it with either tyrosine or isoleucine: both substitutions were nondisruptive, as confirmed by the absence of significant changes in catalytic activity, but altered the tertiary structure (yielding a looser conformation) and decreased the stability towards temperature and denaturants. The change in conformation interferes both with the interaction of the coenzyme to the apoprotein moiety (although the kinetics of the apoprotein-FAD complex reconstitution process are similar between wild-type and mutant D-amino acid oxidases) and with the interaction between monomers. Our results indicate that, in the folded holoenzyme, Trp243 is situated at a position optimal for increasing the interactions between monomers by maximizing van der Waals interactions and by efficiently excluding solvent.

D-Amino-Acid Oxidase↗

PubMed2005

Evolution of an acylase active on cephalosporin C.

Semisynthetic cephalosporins are synthesized from 7-amino cephalosporanic acid, which is produced by chemical deacylation or by a two-step enzymatic process of the natural antibiotic cephalosporin C. The known acylases take glutaryl-7-amino cephalosporanic acid as a primary substrate, and their specificity and activity are too low for cephalosporin C. Starting from a known glutaryl-7-amino cephalosporanic acid acylase as the protein scaffold, an acylase gene optimized for expression in Escherichia coli and for molecular biology manipulations was designed. Subsequently we used error-prone PCR mutagenesis, a molecular modeling approach combined with site-saturation mutagenesis, and site-directed mutagenesis to produce enzymes with a cephalosporin C/glutaryl-7-amino cephalosporanic acid catalytic efficiency that was increased up to 100-fold, and with a significant and higher maximal activity on cephalosporin C as compared to glutaryl-7-amino cephalosporanic acid (e.g., 3.8 vs. 2.7 U/mg protein, respectively, for the A215Y-H296S-H309S mutant). Our data in a bioreactor indicate an ~90% conversion of cephalosporin C to 7-amino-cephalosporanic acid in a single deacylation step. The evolved acylase variants we produced are enzymes with a new substrate specificity, not found in nature, and represent a hallmark for industrial production of 7-amino cephalosporanic acid.

Amidohydrolases↗

PubMed2005

Expression in Escherichia coli and in vitro refolding of the human protein pLG72.

Recently, genes coding for pLG72 and d-amino acid oxidase have been related to schizophrenia, a widespread psychiatric disorder that affects about 1% of population. pLG72 is a puzzling, novel protein present only in primates and proposed to be an activator of d-amino acid oxidase. Here we report on the overexpression of wild-type and His-tagged pLG72 in Escherichia coli. Both variants form inclusion bodies and have been refolded and purified to homogeneity: the acquisition of secondary and tertiary structure was demonstrated by CD spectroscopy. A figure of approximately 70 mg of pure protein per liter of fermentation broth was achieved.

Amino Acid Sequence↗

PubMed2005

Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin.

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His(69) residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (approximately 3 versus approximately 5 M, respectively), and the mutant protein had a melting temperature approximately 10-15 degrees C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 m urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.

Anilino Naphthalenesulfonates↗

PubMed2005

Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine.

The gliotransmitter D-serine is released upon (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and metabotropic glutamate receptor stimulation, but the mechanisms involved are unknown. Here, by using a highly sensitive bioassay to continuously monitor extracellular D-serine levels, we have investigated the pathways used in its release. We reveal that D-serine release is inhibited by removal of extracellular calcium and augmented by increasing extracellular calcium or after treatment with the Ca(2+) ionophore A23187. Furthermore, release of the amino acid is considerably reduced after depletion of thapsigargin-sensitive intracellular Ca(2+) stores or chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester. Interestingly, D-serine release also was markedly reduced by concanamycin A, a vacuolar-type H(+)-ATPase inhibitor, indicating a role for the vesicular proton gradient in the transmitter storage/release. In addition, agonist-evoked D-serine release was sensitive to tetanus neurotoxin. Finally, immunocytochemical and sucrose density gradient analysis revealed that a large fraction of D-serine colocalized with synaptobrevin/VAMP2, suggesting that it is stored in VAMP2-bearing vesicles. In summary, our study reveals the cellular mechanisms subserving D-serine release and highlights the importance of the glial cell exocytotic pathway in influencing CNS levels of extracellular D-serine.

Animals↗

PubMed2004

On the mechanism of Rhodotorula gracilis D-amino acid oxidase: role of the active site serine 335.

Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.

Base Sequence↗

PubMed2004

Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution.

Recent research on the flavoenzyme D-amino acid oxidase from Rhodotorula gracilis (RgDAAO) has revealed new, intriguing properties of this catalyst and offers novel biotechnological applications. Among them, the reaction of RgDAAO has been exploited in the analytical determination of the D-amino acid content in biological samples. However, because the enzyme does not oxidize acidic D-amino acids, it cannot be used to detect the total amount of D-amino acids. We now present the results obtained using a random mutagenesis approach to produce RgDAAO mutants with a broader substrate specificity. The libraries of RgDAAO mutants were generated by error-prone PCR, expressed in BL21(DE3)pLysS Escherichia coli cells and screened for their ability to oxidize different substrates by means of an activity assay. Five random mutants that have a 'modified' substrate specificity, more useful for the analytical determination of the entire content of D-amino acids than wild-type RgDAAO, have been isolated. With the only exception of Y223 and G199, none of the effective amino acid substitutions lie in segments predicted to interact directly with the bound substrate. The substitutions appear to cluster on the protein surface: it would not have been possible to predict that these substitutions would enhance DAAO activity. We can only conclude that these substitutions synergistically generate small structural changes that affect the dynamics and/or stability of the protein in a way that enhances substrate binding or subsequently catalytic turnover.

Alanine↗

PubMed2004

Structure-function correlation in glycine oxidase from Bacillus subtilis.

Structure-function relationships of the flavoprotein glycine oxidase (GO), which was recently proposed as the first enzyme in the biosynthesis of thiamine in Bacillus subtilis, has been investigated by a combination of structural and functional studies. The structure of the GO-glycolate complex was determined at 1.8 A, a resolution at which a sketch of the residues involved in FAD binding and in substrate interaction can be depicted. GO can be considered a member of the "amine oxidase" class of flavoproteins, such as d-amino acid oxidase and monomeric sarcosine oxidase. With the obtained model of GO the monomer-monomer interactions can be analyzed in detail, thus explaining the structural basis of the stable tetrameric oligomerization state of GO, which is unique for the GR(2) subfamily of flavooxidases. On the other hand, the three-dimensional structure of GO and the functional experiments do not provide the functional significance of such an oligomerization state; GO does not show an allosteric behavior. The results do not clarify the metabolic role of this enzyme in B. subtilis; the broad substrate specificity of GO cannot be correlated with the inferred function in thiamine biosynthesis, and the structure does not show how GO could interact with ThiS, the following enzyme in thiamine biosynthesis. However, they do let a general catabolic role of this enzyme on primary or secondary amines to be excluded because the expression of GO is not inducible by glycine, sarcosine, or d-alanine as carbon or nitrogen sources.

Alcohols↗

PubMed2004

Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase.

The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the beta-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal alpha-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal betaalphabeta flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.

Amino Acid Motifs↗

PubMed2003

Regulation of D-amino acid oxidase expression in the yeast Rhodotorula gracilis.

Rhodotorula gracilis is a oleaginous yeast which utilizes D-amino acids as a source of carbon and/or nitrogen. D-amino acid oxidase (DAAO), which converts D-amino acids in the corresponding alpha-keto acids and ammonia, is the first enzyme involved in the catabolism of D-amino acids. DAAO activity is induced by the presence of D-alanine, but the presence of the L-isomer prevents induction by inhibiting the transport of D-alanine into cells. To understand how DAAO expression is regulated, R. gracilis cells were grown on media containing different nitrogen and/or carbon sources. As a general rule, the level of DAAO mRNA reached a maximum after 15 h growth and preceded by approximately 6 h the maximum level of DAAO activity. The inducer D-alanine acts by increasing the rate of DAAO mRNA transcription: the increase in DAAO expression is due essentially to de novo synthesis. The presence of a supplemental carbon source (e.g. succinate or glucose) does not repress DAAO expression. Ammonium sulphate appears to have a negative effect on DAAO mRNA translation and on the expression of DAAO activity: DAAO is only partially active when the yeast is grown in the presence of D-alanine and ammonium sulphate. The best expression of DAAO activity was obtained by growing the cells for 12 h at 30 degrees C in the presence of glucose and D-alanine using cells pre-cultured for 10 h on glucose and L-alanine (0.99 U/mg protein, corresponding to approximately 1.0% total proteins in the crude extract). Under these growth conditions a six-fold increase in DAAO production was achieved.

Alanine↗

PubMed2003

Kinetic mechanisms of glycine oxidase from Bacillus subtilis.

The kinetic properties of glycine oxidase from Bacillus subtilis were investigated using glycine, sarcosine, and d-proline as substrate. The turnover numbers at saturating substrate and oxygen concentrations were 4.0 s(-1), 4.2 s(-1), and 3.5 s(-1), respectively, with glycine, sarcosine, and D-proline as substrate. Glycine oxidase was converted to a two-electron reduced form upon anaerobic reduction with the individual substrates and its reductive half-reaction was demonstrated to be reversible. The rates of flavin reduction extrapolated to saturating substrate concentration, and under anaerobic conditions, were 166 s(-1), 170 s(-1), and 26 s(-1), respectively, with glycine, sarcosine, and D-proline as substrate. The rate of reoxidation of reduced glycine oxidase with oxygen in the absence of product (extrapolated rate approximately 3 x 10(4) M(-1) x s(-1)) was too slow to account for catalysis and thus reoxidation started from the reduced enzyme:imino acid complex. The kinetic data are compatible with a ternary complex sequential mechanism in which the rate of product dissociation from the reoxidized enzyme form represents the rate-limiting step. Although glycine oxidase and D-amino acid oxidase differ in substrate specificity and amino acid sequence, the kinetic mechanism of glycine oxidase is similar to that determined for mammalian D-amino acid oxidase on neutral D-amino acids, further supporting a close similarity between these two amine oxidases.

Amino Acid Oxidoreductases↗

PubMed2003

Dissection of the structural determinants involved in formation of the dimeric form of D-amino acid oxidase from Rhodotorula gracilis: role of the size of the betaF5-betaF6 loop.

The role of the long loop connecting beta-strands F5 and F6 (21 amino acids, Pro302-Leu-Asp-Arg-Thr-Lys-Ser-Pro-Leu-Ser-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu322) present in Rhodotorula gracilis d-amino acid oxidase (RgDAAO) was investigated by site-directed mutagenesis. This loop was proposed to play an important role in the 'head-to-tail' monomer-monomer interaction of this dimeric flavoenzyme: in particular, by means of electrostatic interactions between positively charged residues of the betaF5-betaF6 loop of one monomer and negatively charged residues belonging to the alpha-helices I3' and I3" of the other monomer. We produced a mutant of RgDAAO (namely, DAAO-DeltaLOOP2), in which only minor structural perturbations were introduced (only five amino acids were deleted; new sequence of the betaF5-betaF6 loop is Pro302-Leu-Asp-Arg-Thr-Leu-Gly-Arg-Gly-Ser-Ala-Arg-Ala-Ala-Lys-Glu317), and the charge of the betaF5-betaF6 loop not modified. The DeltaLOOP2 mutant is monomeric, has a weaker binding with the FAD cofactor, a decrease of the kinetic efficiency, and slight modifications in its spectral properties. The short version of the loop does not allow a correct monomer-monomer interaction, and its presence in the monomeric DAAO is a destabilizing structural element since the DeltaLOOP2 mutant is highly susceptible to proteolysis. These results, confirming the role of this loop in the subunits interaction and thus in stabilization of the sole dimeric form of RgDAAO, put forward the evidence that even a short deletion of the loop generates a consistent variation of the enzyme structure-function properties.

D-Amino-Acid Oxidase↗

PubMed2003

Contribution of the dimeric state to the thermal stability of the flavoprotein D-amino acid oxidase.

The flavoenzyme DAAO from Rhodotorula gracilis, a structural paradigm of the glutathione-reductase family of flavoproteins, is a stable homodimer with a flavin adenine dinucleotide (FAD) molecule tightly bound to each 40-kD subunit. In this work, the thermal unfolding of dimeric DAAO was compared with that of two monomeric forms of the same protein: a Deltaloop mutant, in which 14 residues belonging to a loop connecting strands betaF5-betaF6 have been deleted, and a monomer obtained by treating the native holoenzyme with 0.5 M NH(4)SCN. Thiocyanate specifically and reversibly affects monomer association in wild-type DAAO by acting on hydrophobic residues and on ionic pairs between the betaF5-betaF6 loop of one monomer and the alphaI3' and alphaI3" helices of the symmetry-related monomer. By using circular dichroism spectroscopy, protein and flavin fluorescence, activity assays, and DSC, we demonstrated that thermal unfolding involves (in order of increasing temperatures) loss of tertiary structure, followed by loss of some elements of secondary structure, and by general unfolding of the protein structure that was concomitant to FAD release. Temperature stability of wild-type DAAO is related to the presence of a dimeric structure that affects the stability of independent structural domains. The monomeric Deltaloop mutant is thermodynamically less stable than dimeric wild-type DAAO (with melting temperatures (T(m)s) of 48 degrees C and 54 degrees C, respectively). The absence of complications ensuing from association equilibria in the mutant Deltaloop DAAO allowed identification of two energetic domains: a low-temperature energetic domain related to unfolding of tertiary structure, and a high-temperature energetic domain related to loss of secondary structure elements and to flavin release.

D-Amino-Acid Oxidase↗

PubMed2002

Yeast D-amino acid oxidase: structural basis of its catalytic properties.

The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9A) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF(3)-alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2-C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.

Catalytic Domain↗

PubMed2002

Conversion of the dimeric D-amino acid oxidase from Rhodotorula gracilis to a monomeric form. A rational mutagenesis approach.

The relevance of the dimeric state for the structure/function relationships of Rhodotorula gracilis D-amino acid oxidase (RgDAAO) holoenzyme has been investigated by rational mutagenesis. Deletion of 14 amino acids in a surface loop (connecting beta-strands 12 and 13) transforms RgDAAO from a dimeric protein into a stable monomer. The mutant enzyme is still catalytically competent and retains its binding with the FAD coenzyme. Dimerization has been used by this flavoenzyme in evolution to achieve maximal activity, a tighter interaction between the protein moiety and the coenzyme, and higher thermal stability.

Amino Acid Sequence↗