Relative hepatotoxicity of ortho and meta mono substituted thiobenzamides in the rat

Thiobenzamide is known to be hepatotoxic in the rat and the relative hepatotoxicity of para-substituted thiobenzamides has previously been shown to depend strictly on the electronic character of the para substituent. We have now extended this study to include ortho and meta monosubstituted thiobenzamides. Among the meta-substituted compounds, hepatotoxicity varies in strict accordance with the electronic character of the substituent, whereas the ortho-substituted compounds show no toxicity at comparable doses regardless of the nature of the substituent. Explanations for these substituent effects are provided in terms of the chemical reactivity of the compounds and their corresponding S-oxide and S,S-dioxide metabolites.     

Biotransformation of organic sulfides: Part. 10. Formation of chiral ortho- and meta-substituted benzyl methyl sulfoxides by biotransformation using Helminthosporium species NRRL 4671

Benzyl methyl sulfides substituted with methyl, chloro, cyano, bromo, methoxy, nitro and amino groups in the ortho or meta positions of the aromatic ring have been converted to (S) sulfoxides by biotransformation using the fungal biocatalyst Helminthosporium species NRRL 4671. The enantiomeric excesses for meta-substituted examples were high in those cases where the substituent was of a polar nature, and comparable to those observed for the corresponding para-substituted substrates. With one exception (o-amino), the ortho-substituted examples gave sulfoxides of lower enantiomeric purity. The role of a suitably located polar substituent on an aryl ring of the substrate in ensuring a high enantiomeric excess in sulfoxidation by Helminthosporium species has been confirmed by the biotransformations of 4-(methylthiomethyl)benzyl alcohol and 2-(4-nitrophenyl) ethyl methyl sulfide, which give sulfoxides of much higher optical purity than those obtained from the corresponding unsubstituted substrates.     

Isolation and characterization of Xenopus soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.     

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.     

Unravelling interactions between active site residues and DMAP in the initial steps of prenylated flavin mononucleotide biosynthesis catalyzed by PaUbiX

BackgroundPrenylated flavin mononucleotide (prFMN) is a recently discovered, heavily modified flavin compound. It is the only known cofactor that enables enzymatic 1,3-dipolar cycloaddition reactions. It is produced by enzymes from the UbiX family, from flavin mononucleotide and either dimethylallyl mono- or diphosphate. prFMN biosynthesis is currently reported to be initiated by protonation of the substrate by Glu140.MethodsComputational chemistry methods are applied herein - Constant pH MD, classical MD simulations, and QM cluster optimizations.ResultsGlu140 competes for a single proton with Lys129 prior to prFMN biosynthesis, but it is the latter that adopted a protonated state. Once the prenyl-FMN adduct is formed, Glu140 occurs in a protonated state far more often, while the occupancy of protonated Lys129 does not change. Lys129, Glu140, and Arg122 seem to play a key role in either stabilizing or protonating DMAP phosphate group within the PaUbiX active site throughout initial steps of prFMN biosynthesis.ConclusionsThe role of Lys129 in the functioning of PaUbiX is reported for the first time. Glu140 is unlikely to act as a proton donor in prFMN biosynthesis. Instead, Lys129 and Arg122 fulfil this role. Glu140 still plays a role in contributing to hydrogen-bond network. This behavior is most likely conserved throughout the UbiX family due to the structural similarity of the active sites of those proteins.SignificanceMechanistic insights into a crucial biochemical process, the biosynthesis of prFMN, are provided. This study, although purely computational, extends and perfectly complements the knowledge obtained in classical laboratory experiments.     

Effects of Lys and His supplementations on the regulation of nitrogen metabolism in lager yeast

Significant positive correlations between wort fermentability and the assimilation of Lys and His under normal-gravity and high-gravity conditions indicated that Lys and His were the key amino acids for lager yeast during beer brewing. In order to obtain insight into the roles of Lys and His in nitrogen regulation, the influences of Lys, His and their mixture supplementations on the fermentation performance and nitrogen metabolism in lager yeast during high-gravity fermentation were further investigated in the present study. Results showed that Lys and His supplementations improved yeast growth, wort fermentability, ethanol yield and the formation of flavor volatiles. Lys supplementation up-regulated Ssy1p–Ptr3p–Ssy5p (SPS)-regulated genes (LYP1, HIP1, BAP2 and AGP1) dramatically compared to nitrogen catabolite repression (NCR)-sensitive genes (GAP1 and MEP2), whereas His supplementation activated SPS-regulated genes slightly in exponential phase, and repressed NCR-sensitive genes significantly throughout the fermentation. Lys and His supplementations increased the consumption of Glu and Phe, and decreased the consumption of Ser, Trp and Arg. Moreover, Lys and His supplementations exhibited similar effects on the fermentation performance, and were more effective than their mixture supplementation when the same dose was kept. These results demonstrate that both Lys and His are important amino acids for yeast nitrogen metabolism and fermentation performance.     

Benzoyl Coenzyme A Pathway-Mediated Metabolism of meta-Hydroxy-Aromatic Acids in Rhodopseudomonas palustris

Photoheterotrophic metabolism of two meta-hydroxy-aromatic acids, meta-, para-dihydroxybenzoate (protocatechuate) and meta-hydroxybenzoate, was investigated in Rhodopseudomonas palustris. When protocatechuate was the sole organic carbon source, photoheterotrophic growth in R. palustris was slow relative to cells using compounds known to be metabolized by the benzoyl coenzyme A (benzoyl-CoA) pathway. R. palustris was unable to grow when meta-hydroxybenzoate was provided as a sole source of organic carbon under photoheterotrophic growth conditions. However, in cultures supplemented with known benzoyl-CoA pathway inducers (para-hydroxybenzoate, benzoate, or cyclohexanoate), protocatechuate and meta-hydroxybenzoate were taken up from the culture medium. Further, protocatechuate and meta-hydroxybenzoate were each removed from cultures containing both meta-hydroxy-aromatic acids at equimolar concentrations in the absence of other organic compounds. Analysis of changes in culture optical density and in the concentration of soluble organic compounds indicated that the loss of these meta-hydroxy-aromatic acids was accompanied by biomass production. Additional experiments with defined mutants demonstrated that enzymes known to participate in the dehydroxylation of para-hydroxybenzoyl-CoA (HbaBCD) and reductive dearomatization of benzoyl-CoA (BadDEFG) were required for metabolism of protocatechuate and meta-hydroxybenzoate. These findings indicate that, under photoheterotrophic growth conditions, R. palustris can degrade meta-hydroxy-aromatic acids via the benzoyl-CoA pathway, apparently due to the promiscuity of the enzymes involved.     

An evolutionally conserved Lys122 is essential for function in Rhodospirillum rubrum bona fide RuBisCO and Bacillus subtilis RuBisCO-like protein

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and RuBisCO-like protein (RLP) catalyze similar enolase-type reactions. Both enzymes have a conserved non-catalytic Lys122 or Arg122 on the β-strand E lying in the interface between the N- and C-terminal domains. We used site-directed mutagenesis to analyze the function of Lys122 in the form II Rhodospirillum rubrum RuBisCO (RrRuBisCO) and Bacillus subtilis RLP (BsRLP). The K122R mutant of RrRuBisCO had a 40% decrease in k_cat for carboxylase activity, a 2-fold increase in K_m for CO₂, and a 1.9-fold increase in K_m for ribulose-1,5-bisphosphate. K122M and K122E mutants of RrRuBisCO were almost inactive. None of the substitutions affected the thermal stability of RrRuBisCO. The K122R mutant of BsRLP had a 32% decrease in k_cat and lower thermal stability than the wild-type enzyme. The K122M and K122E mutants of BsRLP failed to form a catalytic dimer. Our results suggest that the lysine residue is essential for function in both enzymes, although in each case, its role is likely distinct.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Lysine can replace arginine 67 in the mediation of covalent attachment of FAD to histidine 71 of 6-hydroxy-D-nicotine oxidase

The requirements for FAD-attachment to His71 of 6-hydroxy-D-nicotine oxidase (6-HDNO) were investigated by site-directed mutagenesis. The following amino acid replacements were introduced into the sequence Arg67-Ser68-Gly69-Gly70-His71 of the 6-HDNO-polypeptide: 1) Arg67 was replaced with Ala (A1 mutant); 2) Ser68 was replaced with Ala (A2 mutant); and 3) Arg67 was replaced with Lys (K mutant). The substitution in mutant A2 had no effect on flavinylation, measured as [14C]FAD incorporation into apo-6-HDNO. Replacement of Arg67 with Ala prevented, but replacement with Lys permitted the flavinylation of His71. Mutant A1 showed no 6-HDNO activity, whereas the replacement of Ser with Ala in mutant A2 had only a slight effect on 6-HDNO activity. The substitution of Lys for Arg67, however, reduced the specific 6-HDNO activity in extracts of Escherichia coli cells expressing the mutant polypeptide from 50.3 to 17.5 milliunits/mg protein. It is concluded that a basic amino acid residue (Arg67 or Lys67) is required to mediate the attachment of FAD to His71, and while Lys can substitute for Arg67 in this function, it can only partially replace Arg67 in the enzyme reaction mechanism of 6-HDNO.     

The molecular basis of aminoacylase 1 deficiency

Aminoacylase 1 is a zinc-binding enzyme which hydrolyzes N-acetyl amino acids into the free amino acid and acetic acid. Deficiency of aminoacylase 1 due to mutations in the aminoacylase 1 (ACY1) gene follows an autosomal-recessive trait of inheritance and is characterized by accumulation of N-acetyl amino acids in the urine. In affected individuals neurological findings such as febrile seizures, delay of psychomotor development and moderate mental retardation have been reported. Except for one missense mutation which has been studied in Escherichia coli, mutations underlying aminoacylase 1 deficiency have not been characterized so far. This has prompted us to approach expression studies of all mutations known to occur in aminoacylase 1 deficient individuals in a human cell line (HEK293), thus providing the authentic human machinery for posttranslational modifications. Mutations were inserted using site directed mutagenesis and aminoacylase 1 enzyme activity was assessed in cells overexpressing aminoacylase 1, using mainly the natural high affinity substrate N-acetyl methionine. Overexpression of the wild type enzyme in HEK293 cells resulted in an approximately 50-fold increase of the aminoacylase 1 activity of homogenized cells. Most mutations resulted in a nearly complete loss of enzyme function. Notably, the two newly discovered mutations p.Arg378Trp, p.Arg378Gln and the mutation p.Arg393His yielded considerable residual activity of the enzyme, which is tentatively explained by their intramolecular localization and molecular characteristics. In contrast to aminoacylase 1 variants which showed no detectable aminoacylase 1 activity, aminoacylase 1 proteins with the mutations p.Arg378Trp, p.Arg378Gln and p.Arg393His were also detected in Western blot analysis. Investigations of the molecular bases of additional cases of aminoacylase 1 deficiency contribute to a better understanding of this inborn error of metabolism whose clinical significance and long-term consequences remain to be elucidated.     

Alkenyl substituted bicyclic nucleoside analogues retain nanomolar potency against Varicella Zoster Virus

Novel alkenyl substituted aryl bicyclic furano pyrimidines have been prepared and evaluated in vitro against Varicella Zoster Virus (VZV). The para-substituted analogues retain the nanomolar potency we have reported for p-alkyl analogues, while the ortho- and meta-alkenyl systems lose 3–4 orders of potency.     

Crystal structure of the ribonuclease P protein Ph1877p from hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of pre-tRNA. Protein Ph1877p is one of essential components of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 RNase P [Biochem. Biophys. Res. Commun. 306 (2003) 666]. The crystal structure of Ph1877p was determined at 1.8A by X-ray crystallography and refined to a crystallographic R factor of 22.96% (Rfree of 26.77%). Ph1877p forms a TIM barrel structure, consisting of ten alpha-helices and seven beta-strands, and has the closest similarity to the TIM barrel domain of Escherichia coli cytosine deaminase with a root-mean square deviation of 3.0A. The protein Ph1877p forms an oblate ellipsoid, approximate dimensions being 45Ax43Ax39A, and the electrostatic representation indicated the presence of several clusters of positively charged amino acids present on the molecular surface. We made use of site-directed mutagenesis to assess the role of twelve charged amino acids, Lys42, Arg68, Arg87, Arg90, Asp98, Arg107, His114, Lys123, Lys158, Arg176, Asp180, and Lys196 related to the RNase P activity. Individual mutations of Arg90, Arg107, Lys123, Arg176, and Lys196 by Ala resulted in reconstituted particles with reduced enzymatic activities (32-48%) as compared with that reconstituted RNase P by wild-type Ph1877p. The results presented here provide an initial step for definite understanding of how archaeal and eukaryotic RNase Ps mediate substrate recognition and process 5'-leader sequence of pre-tRNA.     

Crystal Structure of the Light-Driven Chloride Pump Halorhodopsin from Natronomonas pharaonis

The light-driven chloride pump halorhodopsin from Natronomonas pharaonis (phR) crystallised into the monoclinic space group C2, with a phR trimer per the asymmetric unit. Diffraction data at 2.0-Å resolution showed that the carotenoid bacterioruberin binds to crevices between adjacent protein subunits in the trimeric assembly. Besides seven transmembrane helices (A to G) that characterise archaeal rhodopsins, the phR protomer possesses an amphipathic α-helix (A′) at the N-terminus. This helix, together with a long loop between helices B and C, forms a hydrophobic cap that covers the extracellular surface and prevents a rapid ion exchange between the active centre and the extracellular medium. The retinal bound to Lys256 in helix G takes on an all-trans configuration with the Schiff base being hydrogen-bonded to a water molecule. The Schiff base also interacts with Asp252 and a chloride ion, the latter being fixed by two polar groups (Thr126 and Ser130) in helix C. In the anion uptake pathway, four ionisable residues (Arg123, Glu234, Arg176 and His100) and seven water molecules are aligned to form a long hydrogen-bonding network. Conversely, the cytoplasmic half is filled mostly by hydrophobic residues, forming a large energetic barrier against the transport of anion. The height of this barrier would be lowered substantially if the cytoplasmic half functions as a proton/HCl antiporter. Interestingly, there is a long cavity extending from the main-chain carbonyl of Lys256 to Thr71 in helix B. This cavity, which is commonly seen in halobacterial light-driven proton pumps, is one possible pathway that is utilised for a water-mediated proton transfer from the cytoplasmic medium to the anion, which is relocated to the cytoplasmic channel during the photocycle.     

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg²⁺, Ca²⁺ and Ba²⁺) and anions (SO₄²⁻ and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.     

Structure of N-acetyl-l-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor l-arginine bound

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in l-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by l-arginine, although l-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l-arginine, we have determined the structure of the mmNAGS/K complexed with l-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of l-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.     

Identification of active-site amino acid residues in the Chiba virus 3C-like protease

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.     

Prelog and anti-Prelog stereoselectivity of two ketoreductases from Candida glabrata

Two ketoreductases from Candida glabrata were used for the asymmetric reduction of prochiral substituted acetophenones displayed different enantiopreference toward para-, meta-substituted and ortho-halogen substituted acetophenones with excellent enantioselectivity. Homology modeling and docking analysis were in conformity with this interested enantiopreference obtained from experimental tests. The reduction of a series of other substituted aryl ketones was also investigated using the two ketoreductases.