Engineering and Characterization of an Enzyme Replacement Therapy for Classical Homocystinuria

Homocystinuria due to loss of cystathionine beta-synthase (CBS) causes accumulation of homocysteine and depletion of cysteine. Current treatments are suboptimal, and thus the development of an enzyme replacement therapy based on PEGylated human truncated CBS (PEG-CBS) has been initiated. Attenuation of potency was observed, which necessitated a screen of several PEG-CBS conjugates for their efficacy to correct and maintain the plasma metabolite profile of murine homocystinuria after repeated administrations interrupted with washouts. We found that CBS coupling with maleimide PEG inconsistently modified the enzyme. In contrast, the PEG-CBS conjugate with 20 kDa <i>N</i>-hydroxysuccinimide-PEG showed very little loss of potency likely due to a reproducible PEGylation resulting in species modified with five PEGs per subunit on average. We developed assays suitable for monitoring the extent of CBS PEGylation and demonstrated a sustainable partial normalization of homocystinuria upon continuous PEG-CBS administration via osmotic pumps. Taken together, we identified the PEG-CBS conjugate suitable for manufacturing and clinical development

Cross-Talk between the Catalytic Core and the Regulatory Domain in Cystathionine β-Synthase: Study by Differential Covalent Labeling and Computational Modeling

Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by <i>S</i>-adenosyl-l-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conformational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chain reactivity of the truncated CBS lacking the regulatory domain (45CBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms

Cross-Talk between the Catalytic Core and the Regulatory Domain in Cystathionine β-Synthase: Study by Differential Covalent Labeling and Computational Modeling

Cystathionine β-synthase (CBS) is a modular enzyme which catalyzes condensation of serine with homocysteine. Cross-talk between the catalytic core and the C-terminal regulatory domain modulates the enzyme activity. The regulatory domain imposes an autoinhibition action that is alleviated by <i>S</i>-adenosyl-l-methionine (AdoMet) binding, by deletion of the C-terminal regulatory module, or by thermal activation. The atomic mechanisms of the CBS allostery have not yet been sufficiently explained. Using pulse proteolysis in urea gradient and proteolytic kinetics with thermolysin under native conditions, we demonstrated that autoinhibition is associated with changes in conformational stability and with sterical hindrance of the catalytic core. To determine the contact area between the catalytic core and the autoinhibitory module of the CBS protein, we compared side-chain reactivity of the truncated CBS lacking the regulatory domain (45CBS) and of the full-length enzyme (wtCBS) using covalent labeling by six different modification agents and subsequent mass spectrometry. Fifty modification sites were identified in 45CBS, and four of them were not labeled in wtCBS. One differentially reactive site (cluster W408/W409/W410) is a part of the linker between the domains. The other three residues (K172 and/or K177, R336, and K384) are located in the same region of the 45CBS crystal structure; computational modeling showed that these amino acid side chains potentially form a regulatory interface in CBS protein. Subtle differences at CBS surface indicate that enzyme activity is not regulated by conformational conversions but more likely by different allosteric mechanisms

Removal of the regulatory C-terminal domain in dCBS and yCBS.

<p>Four and three STOP codons were introduced to the linker region of dCBS (A) and yCBS (B), respectively, in order to prepare the truncated enzymes. 25 ug of soluble clarified bacterial crude extract (left panels) or solubilized denatured insoluble fraction (right panels) were loaded per lane and separated on 9% SDS-PAGE gels, transferred to a PVDF membrane and probed with either monoclonal anti-6xHis antibody (ABM) (for dCBS) or monoclonal anti-GST antibody (ABM) (for yCBS). CBS specific activities are shown below the respective lanes of the soluble fractions for each construct.</p

Domain architecture and structure of CBS enzymes and reactions catalyzed by CBS.

<p>(A) Domain architecture of CBS enzymes from <i>H. sapiens</i> (hCBS), <i>D. melanogaster</i> (dCBS) and <i>S. cerevisae</i> (yCBS). Regions corresponding to the central catalytic domain (green) and CBS domain (blue) as well as presence of the cofactors (heme in red, PLP in yellow) are indicated. (B) Crystal structures of dCBS (PDB #3PC2) and hCBSΔ516–525 (PDB #4L0D). Subunits within the dimers are distinguished by red or blue color, while the linker connecting the catalytic domain with the regulatory domain (dashed box) is highlighted in green. Yellow color highlights the residues in the connecting linker, which were targeted by mutagenesis (for more details see <b>Fig. 2</b>). Cofactors, heme and PLP, are shown as sticks. (C) Reactions catalyzed by CBS leading to Cth, H₂S and Cys generation that were characterized in this study.</p

Effect of the Disease-Causing R266K Mutation on the Heme and PLP Environments of Human Cystathionine β‑Synthase

Cystathionine β-synthase (CBS) is an essential pyridoxal 5′-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme <i>b</i> cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe­(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe­(III)-Cys­(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm^–1 downshift in the ν­(Fe–S) resonance. Presence of this longer and weaker Fe­(III)–Cys­(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe–S­(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg²⁶⁶–Cys⁵² interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair

Thermal stability of the purified CBS enzymes.

<p>(A) The effect of thermal pre-treatment of full-length dCBS (squares), yCBS (circles), truncated 45 kDa hCBS (triangles) and yCBS L345* (diamonds) on their specific activities in the canonical reaction. (B) The effect of isothermal incubation (at 37°C) of the truncated 45 kDa hCBS (triangles), full-length dCBS (squares) and yCBS (circles) on their specific activities in the canonical reaction.</p

Sequence alignment of residues constituting the two potential AdoMet binding sites (S1 and S2) in hCBS, dCBS and yCBS.

<p>The residues comprising each region were extracted from a careful analysis of both the amino acid sequences and the crystal structures of dCBS and hCBS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105290#pone.0105290-Koutmos1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105290#pone.0105290-ErenoOrbea1" target="_blank">[33]</a> or 3D model of yCBS. The numbering of secondary elements shown above the sequences was adopted from the crystal structure of hCBS. The location of the conserved aspartate that most typically stabilizes the ribose ring of bound nucleotides in CBS domains is marked with an asterisk.</p

Differential scanning calorimetry (DSC) profiles of the studied CBS enzymes.

<p>DSC thermograms of yCBS (A) and dCBS (B) in the absence and the presence of 100 µM AdoMet (solid lines) overlaid with the best fit curves using a 2-state unfolding model (dashed lines). (C) Illustration of the stabilization effect of AdoMet on hCBS showing the up-shift of the first thermal transition (corresponding to the regulatory domain) by comparing this species to a largely stabilized, well-defined, sharp DSC peak of dCBS. The scan rate was 3°C/min and the protein concentration was 5 µM in protein subunit.</p

Comparative Study of Enzyme Activity and Heme Reactivity in <i>Drosophila melanogaster</i> and <i>Homo sapiens</i> Cystathionine β‑Synthases

Cystathionine β-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway, which is critical for the synthesis of cysteine from methionine in eukaryotes. CBS uses coenzyme pyridoxal 5′-phosphate (PLP) for catalysis, and <i>S</i>-adenosylmethionine regulates the activity of human CBS, but not yeast CBS. Human and fruit fly CBS contain heme; however, the role for heme is not clear. This paper reports biochemical and spectroscopic characterization of CBS from fruit fly <i>Drosophila melanogaster</i> (<i>Dm</i>CBS) and the CO/NO gas binding reactions of <i>Dm</i>CBS and human CBS. Like CBS enzymes from lower organisms (e.g., yeast), <i>Dm</i>CBS is intrinsically highly active and is not regulated by AdoMet. The <i>Dm</i>CBS heme coordination environment, the reactivity, and the accompanying effects on enzyme activity are similar to those of human CBS. The <i>Dm</i>CBS heme bears histidine and cysteine axial ligands, and the enzyme becomes inactive when the cysteine ligand is replaced. The Fe­(II) heme in <i>Dm</i>CBS is less stable than that in human CBS, undergoing more facile reoxidation and ligand exchange. In both CBS proteins, the overall stability of the protein is correlated with the heme oxidation state. Human and <i>Dm</i>CBS Fe­(II) hemes react relatively slowly with CO and NO, and the rate of the CO binding reaction is faster at low pH than at high pH. Together, the results suggest that heme incorporation and AdoMet regulation in CBS are not correlated, possibly providing two independent means for regulating the enzyme