Cloning and expression heterologous alanine dehydrogenase genes: Investigation of reductive amination potential of L-alanine dehydrogenases for green synthesis of alanine derivatives.

Unnatural amino acids (UAAs) offer significant promise in a wide range of applications, including drug discovery, the custom design of peptides and proteins, and their utility and use as markers for monitoring molecular interactions in biological research. The synthesis of UAAs presents a formidable challenge and can be classified into two primary categories: enzymatic and chemical synthesis. Notably, the enzymatic route, specifically asymmetric synthesis, emerges as a an attractive method for procuring enantiopure UAAs with high efficiency, owing to its streamlined and concise reaction mechanism. The current study investigated the reductive amination activity mechanisms of alanine dehydrogenase (L-AlaDH), sourced from a combination of newly and previously characterized microorganisms. Our principal aim was to evaluate the catalytic efficiency of these L-AlaDH enzymes concerning a range of specific ketoacids and pyruvate to ascertain their capability for facilitating the production of both natural and unnatural amino acids. After the characterization processes, mutation points for TtAlaDH were determined and as a result of the mutations, mutants that could use ketocaproate and ketovalerate more effectively than the wild type were obtained. Among the enzymes studied, MetAlaDH exhibited the highest specific activity against pyruvate, 173 U/mg, and a KM value of 1.3 mM. VlAlaDH displayed the most favourable catalytic efficiency with a rate constant of 170 s-1mM-1. On the other hand, AfAlaDH demonstrated the highest catalytic efficiency against α-ketobutyrate (34.0 s-1mM-1) and α-ketovalerate (2.7 s-1mM-1). Of the enzymes investigated in the study, TtAlaDH exhibited the highest effectiveness among bacterial enzymes in catalyzing ketocaproate with a measured catalytic efficiency of about 0.6 s-1mM-1 and a KM value of approximately 0.3 mM. These findings provide valuable insights into the substrate specificity and catalytic performance of L-AlaDHs, enhancing our understanding of their potential applications in various biocatalytic processes.

Application of reductive amination by heterologously expressed Thermomicrobium roseumL-alanine dehydrogenase to synthesize L-alanine derivatives.

Unnatural amino acids are unique building blocks in modern medicinal chemistry as they contain an amino and a carboxylic acid functional group, and a variable side chain. Synthesis of pure unnatural amino acids can be made through chemical modification of natural amino acids or by employing enzymes that can lead to novel molecules used in the manufacture of various pharmaceuticals. The NAD+ -dependent alanine dehydrogenase (AlaDH) enzyme catalyzes the conversion of pyruvate to L-alanine by transferring ammonium in a reversible reductive amination activity. Although AlaDH enzymes have been widely studied in terms of oxidative deamination activity, reductive amination activity studies have been limited to the use of pyruvate as a substrate. The reductive amination potential of heterologously expressed, highly pure Thermomicrobium roseum alanine dehydrogenase (TrAlaDH) was examined with regard to pyruvate, α-ketobutyrate, α-ketovalerate and α-ketocaproate. The biochemical properties were studied, which included the effects of 11 metal ions on enzymatic activity for both reactions. The enzyme accepted both derivatives of L-alanine (in oxidative deamination) and pyruvate (in reductive amination) as substrates. While the kinetic KM values associated with the pyruvate derivatives were similar to pyruvate values, the kinetic kcat values were significantly affected by the side chain increase. In contrast, KM values associated with the derivatives of L-alanine (L-α-aminobutyrate, L-norvaline, and L-norleucine) were approximately two orders of magnitude greater, which would indicate that they bind very poorly in a reactive way to the active site. The modeled enzyme structure revealed differences in the molecular orientation between L-alanine/pyruvate and L-norleucine/α-ketocaproate. The reductive activity observed would indicate that TrAlaDH has potential for the synthesis of pharmaceutically relevant amino acids.

Hepcidin is potential regulator for renin activity.

An association between genetic variants in the genes HFE, HJV, BMP4 and arterial hypertension has been shown earlier. Proteins encoded by these genes participate in the signalling routes leading eventually to the production of the peptide hormone hepcidin. Mutations in these genes have been associated with the abnormal production of hepcidin in the body. This finding led to studies exploring the possible role of hepcidin in regulating the activity of blood pressure related renin-angiotensin system enzymes. We used molecular modelling to find out if it is possible for hepcidin to bind to the active site of the renin-angiotensin system enzymes, especially renin. Fluorometric assays were used to evaluate the inhibitory effect of hepcidin on renin as well as angiotensin converting enzymes 1 and 2. Finally, bio-layer interferometry technique was used to study hepcidin binding to renin. The molecular modelling showed that hepcidin seems to have similar binding properties to the renin active site as angiotensinogen does. Based on fluorometric enzyme activity assay, hepcidin has an inhibitory effect on renin in vitro, too. However, angiotensin converting enzymes 1 and 2 were not inhibited remarkably by hepcidin-25. In bio-layer interferometry analysis hepcidin-renin binding was concentration dependent. Our results suggest that hepcidin could act as an inhibitor to the renin. Nowadays, there is no known biological inhibitor for renin in vivo and our finding may thus have important clinical implications.

The challenges of using NAD+-dependent formate dehydrogenases for CO2 conversion.

In recent years, CO2 reduction and utilization have been proposed as an innovative solution for global warming and the ever-growing energy and raw material demands. In contrast to various classical methods, including chemical, electrochemical, and photochemical methods, enzymatic methods offer a green and sustainable option for CO2 conversion. In addition, enzymatic hydrogenation of CO2 into platform chemicals could be used to produce economically useful hydrogen storage materials, making it a win-win strategy. The thermodynamic and kinetic stability of the CO2 molecule makes its utilization a challenging task. However, Nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs), which have high selectivity and specificity, are attractive catalysts to overcome this issue and convert CO2 into fuels and renewable chemicals. It is necessary to improve the stability, cofactor necessity, and CO2 conversion efficiency of these enzymes, such as by combining them with appropriate hybrid systems. However, metal-independent, NAD+-dependent FDHs, and their CO2 reduction activity have received limited attention to date. This review outlines the CO2 reduction ability of these enzymes as well as their properties, reaction mechanisms, immobilization strategies, and integration with electrochemical and photochemical systems for the production of formic acid or formate. The biotechnological applications of FDH, future perspectives, barriers to CO2 reduction with FDH, and aspects that must be further developed are briefly summarized. We propose that constructing hybrid systems that include NAD+-dependent FDHs is a promising approach to convert CO2 and strengthen the sustainable carbon bio-economy.

Effect of Metal Ions on the Activity of Ten NAD-Dependent Formate Dehydrogenases.

NAD-dependent formate dehydrogenase (FDH) enzymes are frequently used in industrial and scientific applications. FDH is a reversible enzyme that reduces the NAD molecule to NADH and produces CO2 by oxidation of the formate ion, whereas it causes CO2 reduction in the reverse reaction. Some transition metal elements - Fe3+, Mo6+ and W6 + - can be found in the FDH structure of anaerobic and archaeal microorganisms, and these enzymes require cations and other redox-active cofactors for their FDH activity. While NAD-dependent FDHs do not necessarily require any metal cations, the presence of various metal cations can still affect FDH activities. To study the effect of 11 different metal ions, NAD-dependent FDH enzymes from ten different microorganisms were tested: Ancylobacter aquaticus (AaFDH), Candida boidinii (CboFDH), Candida methylica (CmFDH), Ceriporiopsis subvermispora (CsFDH), Chaetomium thermophilum (CtFDH), Moraxella sp. (MsFDH), Myceliophthora thermophila (MtFDH), Paracoccus sp. (PsFDH), Saccharomyces cerevisiae (ScFDH) and Thiobacillus sp. (TsFDH). It was found that metal ions (mainly Cu2+ and Zn2+) could have quite strong inhibition effects on several enzymes in the forward reaction, whereas several cations (Li+, Mg2+, Mn2+, Fe3+ and W6+) could increase the forward reaction of two FDHs. The highest activity increase (1.97 fold) was caused by Fe3+ in AaFDH. The effect on the reverse reaction was minimal. The modelled structures of ten FDHs showed that the active site is formed by 15 highly conserved amino acid residues spatially settling around the formate binding site in a conserved way. However, the residue differences at some of the sites close to the substrate do not explain the activity differences. The active site space is very tight, excluding water molecules, as observed in earlier studies. Structural examination indicated that smaller metal ions might be spaced close to the active site to affect the reaction. Metal ion size showed partial correlation to the effect on inhibition or activation. Affinity of the substrate may also affect the sensitivity to the metal's effect. In addition, amino acid differences on the protein surface may also be important for the metal ion effect.

Engineered formate dehydrogenase from Chaetomium thermophilum, a promising enzymatic solution for biotechnical CO2 fixation.

OBJECTIVES: Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. RESULTS: Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3-. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) compared to that of the wild-type for HCO3- reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. CONCLUSIONS: Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.

Functional effects of active site mutations in NAD+-dependent formate dehydrogenases on transformation of hydrogen carbonate to formate.

Conversion of hydrogen carbonate to formate by mutants of Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) formate dehydrogenases (FDHs) was studied. Hydrogen carbonate is not the primary substrate for the hydride transfer reaction in FDHs. The chosen mutations were selected so that enzyme activity could remain at an adequate level. In CtFDH, the mutation Asn120Cys in the active site inactivated the enzyme for formate (oxidation) but increased the specific activity for hydrogen carbonate (reduction) as a function of substrate concentration. The mutation Asn120Cys in CtFDH increased 6.5-fold the KM, indicating that substrate binding was weakened. A 6.5-fold increase of kcat compensated the lower affinity suggesting that product release was improved. The corresponding mutation Asn119Cys in CmFDH inactivated the enzyme for both substrates. Molecular dynamics simulations indicated that the active site dimensions change differently with different substrates after mutations, and in the mutant Asn120Cys of CtFDH, hydrogen carbonate adopted better reactive position than formate. With hydrogen carbonate, the active site enlarged enough for two hydrogen carbonate molecules to be placed there. The change of Asn119 to bulky Tyr or His in CmFDH requires changes in the active site to accommodate the substrate; activity with formate was retained but not with hydrogen carbonate. This study showed that the active site of FDHs can be modified radically, which gives possibilities for further enzyme engineering to improve the reaction with hydrogen carbonate or carbon dioxide for enzymatic fixing of carbon dioxide.

Chaetomium thermophilum formate dehydrogenase has high activity in the reduction of hydrogen carbonate (HCO3 -) to formate.

While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO-). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3-) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO3- as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO3- to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO3- reduced the reaction rate. CtFDH was modeled in the presence of HCO3- showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3-, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions.

Deciphering metal ion preference and primary coordination sphere robustness of a designed zinc finger with high-resolution mass spectrometry.

Small zinc finger (ZnF) motifs are promising molecular scaffolds for protein design owing to their structural robustness and versatility. Moreover, their characterization provides important insights into protein folding in general. ZnF motifs usually possess an exceptional specificity and high affinity towards Zn(II) ion to drive folding. While the Zn(II) ion is canonically coordinated by two cysteine and two histidine residues, many other coordination spheres also exist in small ZnFs, all having four amino acid ligands. Here we used high-resolution mass spectrometry to study metal ion binding specificity and primary coordination sphere robustness of a designed zinc finger, named MM1. Based on the results, MM1 possesses high specificity for zinc with sub-micromolar binding affinity. Surprisingly, MM1 retains metal ion binding affinity even in the presence of selective alanine mutations of the primary zinc coordinating amino acid residues.

Redox-dependent disulfide bond formation in SAP30L corepressor protein: Implications for structure and function.

Sin3A-associated protein 30-like (SAP30L) is one of the key proteins in a multi-subunit protein complex involved in transcriptional regulation via histone deacetylation. SAP30L, together with a highly homologous SAP30 as well as other SAP proteins (i.e., SAP25, SAP45, SAP130, and SAP180), is an essential component of the Sin3A corepressor complex, although its actual role has remained elusive. SAP30L is thought to function as an important stabilizing and bridging molecule in the complex and to mediate its interactions with other corepressors. SAP30L has been previously shown to contain an N-terminal Cys3 His type zinc finger (ZnF) motif, which is responsible for the key protein-protein, protein-DNA, and protein-lipid interactions. By using high-resolution mass spectrometry, we studied a redox-dependent disulfide bond formation in SAP30L ZnF as a regulatory mechanism for its structure and function. We showed that upon oxidative stress SAP30L undergoes the formation of two specific disulfide bonds, a vicinal Cys29-Cys30 and Cys38-Cys74, with a concomitant release of the coordinated zinc ion. The oxidized protein was shown to remain folded in solution and to bind signaling phospholipids. We also determined a solution NMR structure for SAP30L ZnF that showed an overall fold similar to that of SAP30, determined earlier. The NMR titration experiments with lipids and DNA showed that the binding is mediated by the C-terminal tail as well as both α-helices of SAP30L ZnF. The implications of these results for the structure and function of SAP30L are discussed.

Angiotensin(1-7) and ACE2, "The Hot Spots" of Renin-Angiotensin System, Detected in the Human Aqueous Humor.

BACKGROUND: The main purpose of the study was to establish whether essential components of the renin-angiotensin system (RAS) exist in the human aqueous humor. METHODS: Forty-five patients ≥ 60 (74±7) years of age undergoing cataract surgery at Tampere University Hospital were randomly selected for the prospective study. The exclusion criterion was the use of oral antihypertensive medicine acting via renin-angiotensin system. Aqueous humor samples were taken at the beginning of normal cataract extraction. The samples were frozen and stored at -80 °C. The concentrations of intraocular endogenous RAS components Ang(1-7), ACE2, and ACE1 were measured using ELISA. RESULTS: Concentration medians of Ang(1-7), ACE2, and ACE1 in the aqueous humor were: Ang(1-7) 4.08 ng/ml, ACE2 2.32 ng/ml and ACE1 0.35 ng/ml. The concentrations were significantly higher in glaucomatous than in non-glaucomatous eyes, ACE1 (p=0.014) and Ang(1-7) (p=0.026) vs non-glaucomatous eyes. CONCLUSIONS: Ang(1-7), ACE2 and ACE1 are found in the human aqueous humor. The observations are consistent with the conception that local tissue-RAS exists in the human eye and it might have a role in the control of intraocular pressure.

The talin-integrin interface under mechanical stress.

The major mechanical function of talin is to couple the ß-integrin cytoplasmic tails to actin filaments. A variety of ß-integrin tails contain conserved binding motifs for talin, and recent research shows that ß-integrins differ both in affinity to talin and preferences for other cytoplasmic adaptor proteins. While talin predominantly links ß3 integrins to actin filaments within the peripheral cell adhesion sites, talin can become replaced by other integrin adaptor proteins through their overlapping binding sites on integrin tails. Although the NPxY motif in the ß-integrin tail is important for talin recognition, our simulations suggest considerably smaller contribution of the NPxY motif in the force resistance of the talin-integrin complex than for the residues upstream of the NPxY. It might thus be possible for the NPxY motif to detach from talin and interact with other integrin binding proteins while the ß-integrin still remains bound to talin. The epithelial integrin ß6 reportedly activates latent TGFß1, and we propose that its function may involve direct interaction with talin.

Does the cis/trans configuration of peptide bonds in bioactive tripeptides play a role in ACE-1 enzyme inhibition?

BACKGROUND: The milk casein-derived bioactive tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to prevent development of hypertension in animal models and to lower blood pressure in moderately hypertensive subjects in most but not all clinical trials. Inhibition of angiotensin-converting enzyme 1 (ACE-1) has been suggested as the explanation for these antihypertensive and beneficial vascular effects. Previously, human umbilical vein endothelial cells (HUVEC) have not been used to test ACE-1 inhibiting properties of casein derived tripeptides in vasculature. PURPOSE: We focused on the cis/trans configurations of the peptide bonds in proline-containing tripeptides in order to discover whether the different structural properties of these peptides influence their activity in ACE-1 inhibition. We hypothesized that the configuration of proline-containing peptides plays a significant role in enzyme inhibition. METHODS: AutoDock 4.2 docking software was used to predict suitable peptide bond configurations of the tripeptides. Besides modeling studies, we completed ACE-1 activity measurements in vitro using HUVEC cultures. RESULTS: In HUVEC cells, both IPP and VPP inhibited ACE-1. Based on molecular docking studies, we propose that in ACE-1 inhibition IPP and VPP share a similar cis configuration between the first aliphatic (isoleucine or valine) and the second (proline) amino acid residues and more different configurations between two proline residues. In vivo experiments are needed to validate the significance of the present findings.

Zinc coordination spheres in protein structures.

Zinc metalloproteins are one of the most abundant and structurally diverse proteins in nature. In these proteins, the Zn(II) ion possesses a multifunctional role as it stabilizes the fold of small zinc fingers, catalyzes essential reactions in enzymes of all six classes, or assists in the formation of biological oligomers. Previously, a number of database surveys have been conducted on zinc proteins to gain broader insights into their rich coordination chemistry. However, many of these surveys suffer from severe flaws and misinterpretations or are otherwise limited. To provide a more comprehensive, up-to-date picture on zinc coordination environments in proteins, zinc containing protein structures deposited in the Protein Data Bank (PDB) were analyzed in detail. A statistical analysis in terms of zinc coordinating amino acids, metal-to-ligand bond lengths, coordination number, and structural classification was performed, revealing coordination spheres from classical tetrahedral cysteine/histidine binding sites to more complex binuclear sites with carboxylated lysine residues. According to the results, coordination spheres of hundreds of crystal structures in the PDB could be misinterpreted due to symmetry-related molecules or missing electron densities for ligands. The analysis also revealed increasing average metal-to-ligand bond length as a function of crystallographic resolution, which should be taken into account when interrogating metal ion binding sites. Moreover, one-third of the zinc ions present in crystal structures are artifacts, merely aiding crystal formation and packing with no biological significance. Our analysis provides solid evidence that a minimal stable zinc coordination sphere is made up by four ligands and adopts a tetrahedral coordination geometry.

Structure-function analysis indicates that sumoylation modulates DNA-binding activity of STAT1.

BACKGROUND: STAT1 is an essential transcription factor for interferon-γ-mediated gene responses. A distinct sumoylation consensus site (ψKxE) 702IKTE705 is localized in the C-terminal region of STAT1, where Lys703 is a target for PIAS-induced SUMO modification. Several studies indicate that sumoylation has an inhibitory role on STAT1-mediated gene expression but the molecular mechanisms are not fully understood. RESULTS: Here, we have performed a structural and functional analysis of sumoylation in STAT1. We show that deconjugation of SUMO by SENP1 enhances the transcriptional activity of STAT1, confirming a negative regulatory effect of sumoylation on STAT1 activity. Inspection of molecular model indicated that consensus site is well exposed to SUMO-conjugation in STAT1 homodimer and that the conjugated SUMO moiety is directed towards DNA, thus able to form a sterical hindrance affecting promoter binding of dimeric STAT1. In addition, oligoprecipitation experiments indicated that sumoylation deficient STAT1 E705Q mutant has higher DNA-binding activity on STAT1 responsive gene promoters than wild-type STAT1. Furthermore, sumoylation deficient STAT1 E705Q mutant displayed enhanced histone H4 acetylation on interferon-γ-responsive promoter compared to wild-type STAT1. CONCLUSIONS: Our results suggest that sumoylation participates in regulation of STAT1 responses by modulating DNA-binding properties of STAT1.

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase.

L-Xylulose was used as a raw material for the production of L-xylose with a recombinantly produced Escherichia coli L-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for L-xylulose with a K(m) of 41 mM and a V(max) of 0.23 µmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6h 50 min and 1h 31 min, respectively. The reaction equilibrium between L-xylulose and L-xylose was 15:85 at 35 °C and thus favored the formation of L-xylose. Contrary to the L-rhamnose isomerase catalyzed reaction described previously [14]L-lyxose was not detected in the reaction mixture with L-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to L-xylulose the inhibition did not reach 50%.

Characterization of non-specific cytotoxic cell receptor protein 1: a new member of the lectin-type subfamily of F-box proteins.

Our previous microarray study showed that the non-specific cytotoxic cell receptor protein 1 (Nccrp1) transcript is significantly upregulated in the gastric mucosa of carbonic anhydrase IX (CA IX)-deficient (Car9(-/-)) mice. In this paper, we aimed to characterize human NCCRP1 and to elucidate its relationship to CA IX. Recombinant NCCRP1 protein was expressed in Escherichia coli, and a novel polyclonal antiserum was raised against the purified full-length protein. Immunocytochemistry showed that NCCRP1 is expressed intracellularly, even though it has previously been described as a transmembrane protein. Using bioinformatic analyses, we identified orthologs of NCCRP1 in 35 vertebrate genomes, and up to five paralogs per genome. These paralogs are FBXO genes whose protein products are components of the E3 ubiquitin ligase complexes. NCCRP1 proteins have no signal peptides or transmembrane domains. NCCRP1 has mainly been studied in fish and was thought to be responsible for the cytolytic function of nonspecific cytotoxic cells (NCCs). Our analyses showed that in humans, NCCRP1 mRNA is expressed in tissues containing squamous epithelium, whereas it shows a more ubiquitous tissue expression pattern in mice. Neither human nor mouse NCCRP1 expression is specific to immune tissues. Silencing CA9 using siRNAs did not affect NCCRP1 levels, indicating that its expression is not directly regulated by CA9. Interestingly, silencing NCCRP1 caused a statistically significant decrease in the growth of HeLa cells. These studies provide ample evidence that the current name, "non-specific cytotoxic cell receptor protein 1," is not appropriate. We therefore propose that the gene name be changed to FBXO50.

Acetaldehyde-derived modifications on cytosolic human carbonic anhydrases.

Acetaldehyde can generate modifications in several proteins, such as carbonic anhydrase (CA) II. In this study, we extended in vitro investigations on acetaldehyde adduct formation by focusing on the other human cytosolic CA enzymes I, III, VII, and XIII. High-resolution mass spectrometric analysis indicated that acetaldehyde most efficiently formed covalent adducts with CA II and XIII. The binding of up to 19 acetaldehydes in CA II is probably attributable to the high number of lysine residues (n = 24) located mainly on the surface of the enzyme molecule. CA XIII formed more adducts (up to 25) than it contains lysine residues (n = 16) in its primary structure. Acetaldehyde treatment induced only minor changes in CA catalytic activity in most cases. The present study provides the first evidence that acetaldehyde can bind to several cytosolic CA isozymes. The functional consequences of such modifications will be further investigated in vivo by using animal models.

Analysis of a shortened form of human carbonic anhydrase VII expressed in vitro compared to the full-length enzyme.

Carbonic anhydrase (CA) enzymes are expressed in all organs of the mammalian body where they participate in important physiological functions. CA VII is a cytosolic isozyme which may be expressed as two forms according to the recent GenBank data. We designed a present study to express and characterize the human CA VII forms: full-length CA VII and short form (predicted to lack 56 residues from the N-terminus). Reverse transcriptase PCR analysis revealed mRNAs for both CA VII forms in the human brain. These different forms were expressed as recombinant proteins to investigate their biochemical properties. The full-length CA VII was used to raise a polyclonal antiserum in a rabbit, and the antiserum was then employed in western blot analyses and immunohistochemistry of mouse tissues. Data from mass spectrometry and comparative modeling showed that CA VII protein contains a single intramolecular disulfide bridge (Cys-56 to Cys-180) which is lacking in the short form. The computer model suggested distinctly different folding for the different forms. The more exposed structure and the absence of the disulfide bridge in the short form could make this protein more susceptible to degradation. In fact, this was realized in several protein purification efforts in which the short form readily degraded during the experimental procedures. From these results, we conclude that the full-length CA VII is a predominant active form in human brain and also in other tissues. In addition to the brain, CA VII is expressed in several other organs including the stomach, duodenum, colon, liver, and skeletal muscle. The distribution pattern suggests multiple functions for CA VII in different organs.

Activities of angiotensin-converting enzymes ACE1 and ACE2 and inhibition by bioactive peptides in porcine ocular tissues.

PURPOSE: An active local renin-angiotensin system (RAS) has recently been found in the human eye. The aim of the present study was to compare the activities of central RAS enzymes (ACE1 and 2) in porcine ocular tissues, morphologically and physiologically close to the human eye. In addition, the effects of three ACE-inhibitory tripeptides on these enzymes were evaluated. METHODS: Enucleated fresh porcine eyes were used. Activities of ACE1 and ACE2 and their inhibition by bioactive tripeptides (Ile-Pro-Pro, Val-Pro-Pro, Leu-Pro-Pro) as well as by a standard ACE-inhibitor captopril were assayed in the vitreous body, the retina and the ciliary body using fluorometric detection methods. RESULTS: Activity of ACE1 as well as ACE2 was found in all tissues evaluated. ACE1 activity was markedly higher in the ciliary body (3.7 +/- 0.7 mU/mg protein) than in retina (0.2 +/- 0.02 mU/mg), whereas ACE2 activities in the ciliary body (0.2 +/- 0.02 mU/mg) and retina (0.2 +/- 0.01 mU/mg) were at the same level. In the vitreous body ACE1 activity (8.2 +/- 0.31 nmol/min/mL) was manifold compared to that of ACE2 (0.1 +/- 0.02 nmol/min/mL). The tripeptides inhibited ACE1 at one-thousandth of the concentration needed to inhibit ACE2. All peptides studied evinced about equal inhibitory activities. CONCLUSION: To our knowledge the present findings constitute the first evidence of ACE2 activity in the ciliary and vitreous bodies, in addition to previously described activity in the retina. The known favorable effects of ACE2 products vs. those of ACE1 suggest a counterbalancing interaction of these two enzyme homologues in physiological regulation of ocular circulation and pressure and possible protective role in certain ophthalmic disorders such as glaucoma and diabetic retinopathy.