Characterization of a restriction barrier and electrotransformation of the cyanobacterium Nostoc PCC 7121

We have investigated host restriction as a barrier to transformation and developed a method for gene transfer into the previously untransformable, heterotrophic cyanobacterium Nostoc PCC 7121. A restriction endonuclease, designated Nsp 7121I, has been partially purified by phosphocellulose chromatography of Nostoc cell extract. Comparisons of Nsp 7121I digests of bacteriophage lambda and plasmid DNAs with computer-generated restriction fragment profiles showed that Nsp 7121I is an isoschizomer of restriction endonucleases, such as Asu I, Nsp 7524IV, Sau 96I, and Eco 47II, that recognize the sequence GGNCC. Cleavage by Nsp 7121I within this sequence was confirmed by sequence analysis of DNA fragments cleaved at a unique Nsp 7121I site. These data further suggested that cleavage occurs after the first G (5-G/GNCC-3) in this site to generate a three base 5 overhang. Nsp 7121I degraded all plasmids used in previous transformation attempts but modification of these DNA molecules by Eco 47II methylase effectively prevented digestion by Nsp 7121I. Plasmids premethylated by passage through Escherichia coli carrying a plasmid encoded Eco 47II methylase have now been used in an electroporation procedure to transform Nostoc PCC 7121 to neomycin resistance at frequencies as high as one transformant per 10³ viable cells. Transformation, and stable replication within Nostoc of one of the transforming plasmids (pRL25), was confirmed by recovery of pRL25, in its original form, from transformants. Conjugal transfer of pRL25 from E. coli into Nostoc was also possible but at much lower efficiency than by electroporation. These findings establish the basis for genetic analysis of Nostoc PCC 7121, from which genes for photosynthetic electron transport have been cloned.     

Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

A new Type III restriction endonuclease designated PstII has been purified from Providencia stuartii. PstII recognizes the hexanucleotide sequence 5′-CTGATG(N)_25-26/27-28-3′. Endonuclease activity requires a substrate with two copies of the recognition site in head-to-head repeat and is dependent on a low level of ATP hydrolysis (~40 ATP/site/min). Cleavage occurs at just one of the two sites and results in a staggered cut 25–26 nt downstream of the top strand sequence to generate a two base 5′-protruding end. Methylation of the site occurs on one strand only at the first adenine of 5′-CATCAG-3′. Therefore, PstII has characteristic Type III restriction enzyme activity as exemplified by EcoPI or EcoP15I. Moreover, sequence asymmetry of the PstII recognition site in the T7 genome acts as an historical imprint of Type III restriction activity in vivo. In contrast to other Type I and III enzymes, PstII has a more relaxed nucleotide specificity and can cut DNA with GTP and CTP (but not UTP). We also demonstrate that PstII and EcoP15I cannot interact and cleave a DNA substrate suggesting that Type III enzymes must make specific protein–protein contacts to activate endonuclease activity.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.?coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.     

A physical map of plasmid pDU1 from the cyanobacterium Nostoc PCC 7524

Nostoc 7524 contains three different plasmids of molecular weight, 4, 8, and 28 Mdal. The smallest plasmid, designated pDU1, because of its size and ease of isolation, may prove to be useful as a cloning vector. Plasmid pDU1 was incubated separately with 26 different restriction enzymes and only 8 of the enzymes tested cut pDU1. A composite restriction enzyme map consisting of a total of 17 restriction sites was constructed for BglI, HindIII, HpaI, and XbaI. The sites of restriction enzyme cleavage were determined by single, double, and partial digests of plasmid DNA or redigestion of isolated restriction fragments. All the restriction sites were aligned relative to the single BglI site. This is the first restriction enzyme map of a plasmid from a filamentous cyanobacterium.     

Evidence for horizontal transfer of SsuDAT1I restriction-modification genes to the Streptococcus suis genome

Different strains of Streptococcus suis serotypes 1 and 2 isolated from pigs either contained a restriction-modification (R-M) system or lacked it. The R-M system was an isoschizomer of Streptococcus pneumoniae DpnII, which recognizes nucleotide sequence 5′-GATC-3′. The nucleotide sequencing of the genes encoding the R-M system in S. suis DAT1, designated SsuDAT1I, showed that the SsuDAT1I gene region contained two methyltransferase genes, designated ssuMA and ssuMB, as does the DpnII system. The deduced amino acid sequences of M.SsuMA and M.SsuMB showed 70 and 90% identity to M.DpnII and M.DpnA, respectively. However, the SsuDAT1I system contained two isoschizomeric restriction endonuclease genes, designated ssuRA and ssuRB. The deduced amino acid sequence of R.SsuRA was 49% identical to that of R.DpnII, and R.SsuRB was 72% identical to R.LlaDCHI of Lactococcus lactis subsp. cremoris DCH-4. The four SsuDAT1I genes overlapped and were bounded by purine biosynthetic gene clusters in the following gene order: purF-purM-purN-purH-ssuMA-ssuMB-ssuRA-ssuRB-purD-purE. The G+C content of the SsuDAT1I gene region (34.1%) was lower than that of the pur region (48.9%), suggesting horizontal transfer of the SsuDAT1I system. No transposable element or long-repeat sequence was found in the flanking regions. The SsuDAT1I genes were functional by themselves, as they were individually expressed in Escherichia coli. Comparison of the sequences between strains with and without the R-M system showed that only the region from 53 bp upstream of ssuMA to 5 bp downstream of ssuRB was inserted in the intergenic sequence between purH and purD and that the insertion target site was not the recognition site of SsuDAT1I. No notable substitutions or insertions could be found, and the structures were conserved among all the strains. These results suggest that the SsuDAT1I system could have been integrated into the S. suis chromosome by an illegitimate recombination mechanism.     

Functional Characterization of a Nup159p-containing Nuclear Pore Subcomplex

Nup159p/Rat7p is an essential FG repeat–containing nucleoporin localized at the cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)⁺ RNA export and NPC distribution. A detailed structural–functional analysis of this nucleoporin previously demonstrated that Nup159p is anchored within the NPC through its essential carboxyl-terminal domain. In this study, we demonstrate that Nup159p specifically interacts through this domain with both Nsp1p and Nup82p. Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p subcomplex using the nup82Δ108 mutant strain revealed that a deletion within the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but does not affect the interaction between Nup159p and Nsp1p. Moreover, immunofluorescence analysis demonstrated that Nup159p is delocalized from the NPC in nup82Δ108 cells grown at 37°C, a temperature at which the Nup82Δ108p mutant protein becomes degraded. This suggests that Nup82p may act as a docking site for a core complex composed of the repeat-containing nucleoporins Nup159p and Nsp1p. In vivo transport assays further revealed that nup82Δ108 and nup159-1/rat7-1 mutant strains have little if any defect in nuclear protein import and protein export. Together our data suggest that the poly(A)⁺ RNA export defect previously observed in nup82 mutant cells might be due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.␣coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene. Received: 15 April 1998 / Accepted: 3 August 1998     

Characterization of AbiR, a Novel Multicomponent Abortive Infection Mechanism Encoded by Plasmid pKR223 of Lactococcus lactis subsp. lactis KR2

The native lactococcal plasmid pKR223 encodes two distinct phage resistance mechanisms, a restriction and modification (R/M) system designated LlaKR2I and an abortive infection mechanism (Abi) which affects prolate-headed-phage proliferation. The nucleotide sequence of a 16,174-bp segment of pKR223 encompassing both the R/M and Abi determinants has been determined, and sequence analysis has validated the novelty of the Abi system, which has now been designated AbiR. Analysis of deletion and insertion clones demonstrated that AbiR was encoded by two genetic loci, separated by the LlaKR2I R/M genes. Mechanistic studies on the AbiR phenotype indicated that it was heat sensitive and that it impeded phage DNA replication. These data indicated that AbiR is a novel multicomponent, heat-sensitive, “early”-functioning Abi system and is the first lactococcal Abi system described which is encoded by two separated genetic loci.     

Promiscuous Binding of Karyopherinβ1 Modulates FG Nucleoporin Barrier Function and Expedites NTF2 Transport Kinetics

The transport channel of nuclear pore complexes (NPCs) contains a high density of intrinsically disordered proteins that are rich in phenylalanine-glycine (FG)-repeat motifs (FG Nups). The FG Nups interact promiscuously with various nuclear transport receptors (NTRs), such as karyopherins (Kaps), that mediate the trafficking of nucleocytoplasmic cargoes while also generating a selectively permeable barrier against other macromolecules. Although the binding of NTRs to FG Nups increases molecular crowding in the NPC transport channel, it is unclear how this impacts FG Nup barrier function or the movement of other molecules, such as the Ran importer NTF2. Here, we use surface plasmon resonance to evaluate FG Nup conformation, binding equilibria, and interaction kinetics associated with the multivalent binding of NTF2 and karyopherinβ1 (Kapβ1) to Nsp1p molecular brushes. NTF2 and Kapβ1 show different long- and short-lived binding characteristics that emerge from varying degrees of molecular retention and FG repeat binding avidity within the Nsp1p brush. Physiological concentrations of NTF2 produce a collapse of Nsp1p brushes, whereas Kapβ1 binding generates brush extension. However, the presence of prebound Kapβ1 inhibits Nsp1p brush collapse during NTF2 binding, which is dominated by weak, short-lived interactions that derive from steric hindrance and diminished avidity with Nsp1p. This suggests that binding promiscuity confers kinetic advantages to NTF2 by expediting its facilitated diffusion and reinforces the proposal that Kapβ1 contributes to the integral barrier function of the NPC.     

Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls I : Purification of Novel Enzymes Capable of Dissociating Feraxan Fragments from Zea mays Coleoptile Cell Wall

Three novel β-xylan xylanohydrolases capable of dissociating ferulated arabinoxylan (Feraxan) from maize (Zea mays L. hybrid B73 × Mo17) coleoptile sections and two conventional β-xylan xylanohydrolases (xylanases) were purified from a Bacillus subtilis industrial enzyme preparation (Novo Ban L-120). The Feraxan-dissociating enzymes (designated as feraxanases) exhibit optimum activities between pH 6.5 and 7.0 and have common molecular weights of 45 kilodaltons as studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two xylanases exhibit their optimum activities between pH 4.5 and 6.0 and have common molecular weights of 27 kilodaltons. Feraxanases liberate oligomeric fragments, which accounted for the following percentages of walls of Zea mays coleoptile sections that had been pretreated by boiling in 80% ethanol: 76% of the ferulic acid, 96% of the arabinose, 71% of the xylose, 27% of the galactose, 50% of the uronic acid, and 4% of the glucose. Monomers, dimers, trimers, or tetramers were not found among enzyme digestion products. The enzymes hydrolyzed both Feraxan in intact cell wall and maize arabinoxylans extracted from walls by alkaline solutions but did not degrade other substrates including larch arabinoxylan and Rhodymenia xylan. Structural analyses of the fragments released by the enzymes from the maize cell wall indicated the presence of 2,4/3,4-linked-xylopyranosyl, terminal-arabinofuranosyl, 5-linked-arabinofuranosyl, 4-linked-xylopyranosyl, terminal-glucuronopyranosyl, and ferulic acid as major components. This result is consistent with the idea that most of the fragments were derived from Feraxan. Because of high enzyme specificity and substantial recovery of digestion products from maize cell walls, these new enzymes offer opportunities not only for enhanced structural analyses of cell walls but also for assistance in protoplast preparation from cereals.     

Crystal structure of the Bse634I restriction endonuclease: comparison of two enzymes recognizing the same DNA sequence

Crystal structures of Type II restriction endonucleases demonstrate a conserved common core and active site residues but diverse structural elements involved in DNA sequence discrimination. Comparative structural analysis of restriction enzymes recognizing the same nucleotide sequence might therefore contribute to our understanding of the structural diversity of specificity determinants within restriction enzymes. We have solved the crystal structure of the Bacillus stearothermophilus restriction endonuclease Bse634I by the multiple isomorphous replacement technique to 2.17 Å resolution. Bse634I is an isoschisomer of the Cfr10I restriction enzyme whose crystal structure has been reported previously. Comparative structural analysis of the first pair of isoschisomeric enzymes revealed conserved structural determinants of sequence recognition and catalysis. However, conformations of the N-terminal subdomains differed between Bse634I/Cfr10I, suggesting a rigid body movement that might couple DNA recognition and catalysis. Structural similarities extend to the quaternary structure level: crystal contacts suggest that Bse634I similarly to Cfr10I is arranged as a tetramer. Kinetic analysis reveals that Bse634I is able to interact simultaneously with two recognition sites supporting the tetrameric architecture of the protein. Thus, restriction enzymes Bse634I, Cfr10I and NgoMIV, recognizing overlapping nucleotide sequences, exhibit a conserved tetrameric architecture that is of functional importance.     

Sequence-specific endonucleases in strains of Anabaena and Nostoc

The complements of restriction endonucleases of 12 strains of cyanobacteria were determined in cell-free extracts, and were compared with the complements of restriction activities assessed by measuring the relative efficiencies of plating of cyanophages on those cyanobacteria. The hosts which were susceptible to all of the phages contained endo R · AvaI and endo R · AvaII, and in several cases probably endo R · AvaIII, or isoschizomers of these enzymes. Three hosts which were lysed by only a subset (1 or 3) of the phages contained different restriction endonuclease. Anabaena sp. PCC 7120 showed apparent phenotypic restriction of phage An-22 grown in hosts with (isoschizomers of) AvaI, II and III, but no corresponding endonuclease has yet been detected in vitro. Nostoc sp. ATCC 29131 (PCC 6705) was found to contain a restriction enzyme, NspBII, with hitherot unknown specificity, C(A/C)GC(T/G)G.     

XPF-673C>T Polymorphism Effect on the Susceptibility to Esophageal Cancer in Chinese Population

Purpose

Xeroderma pigmentsum group F (XPF) plays a pivotal role in DNA nucleotide excision repair and has been linked to the development of various cancers. This study aims to assess the association of XPF genetic variants with the susceptibility to esophageal squamous cell carcinoma (ESCC) in Chinese population.

Methods

This two-stage case-control study was conducted in a total of 1524 patients with ESCC and 1524 controls. Genotype of XPF -673C>T and 11985A>G variants were determined by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). Logistic regression analysis was performed to estimate odd ratios (ORs) and 95% confidence intervals (95% CI).

Results

Our case-control study showed that XPF -673TT genotype was associated with a decreased risk of ESCC compared with CC genotype in both case-control sets (Tangshan set: OR = 0.58; 95%CI = 0.34–0.99, P = 0.040; Beijing set: OR = 0.66; 95%CI = 0.46–0.95, P = 0.027). Stratified analyses revealed that a multiplicative interaction between -673C>T variant and age, sex or smoking status was evident (Gene-age: P_interaction = 0.002; Gene-sex: P_interaction = 0.002; Gene-smoking: P_interaction = 0.002). For XPF 11985A>G polymorphism, there was no significant difference of genotype distribution between ESCC cases and controls.

Conclusion

These findings indicated that genetic variants in XPF might contribute to the susceptibility to ESCC.     

Recent insights into noncanonical 5′ capping and decapping of RNA

The 5′ N⁷-methylguanosine cap is a critical modification for mRNAs and many other RNAs in eukaryotic cells. Recent studies have uncovered an RNA 5′ capping quality surveillance mechanism, with DXO/Rai1 decapping enzymes removing incomplete caps and enabling the degradation of the RNAs, in a process we also refer to as “no-cap decay.” It has also been discovered recently that RNAs in eukaryotes, bacteria, and archaea can have noncanonical caps (NCCs), which are mostly derived from metabolites and cofactors such as NAD, FAD, dephospho-CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleotide polyphosphates. These NCCs can affect RNA stability, mitochondrial functions, and possibly mRNA translation. The DXO/Rai1 enzymes and selected Nudix (nucleotide diphosphate linked to X) hydrolases have been shown to remove NCCs from RNAs through their deNADding, deFADding, deCoAping, and related activities, permitting the degradation of the RNAs. In this review, we summarize the recent discoveries made in this exciting new area of RNA biology.     

Mitochondrial Genome Analysis of Primary Open Angle Glaucoma Patients

Primary open angle glaucoma (POAG) is a multi-factorial optic disc neuropathy characterized by accelerating damage of the retinal ganglion cells and atrophy of the optic nerve head. The vulnerability of the optic nerve damage leading to POAG has been postulated to result from oxidative stress and mitochondrial dysfunction. In this study, we investigated the possible involvement of the mitochondrial genomic variants in 101 patients and 71 controls by direct sequencing of the entire mitochondrial genome. The number of variable positions in the mtDNA with respect to the revised Cambridge Reference Sequence (rCRS), have been designated “Segregating Sites”. The segregating sites present only in the patients or controls have been designated “Unique Segregating Sites (USS)”. The population mutation rate (θ = 4N_eμ) as estimated by Watterson’s θ (θ_w), considering only the USS, was significantly higher among the patients (p = 9.8×10⁻¹⁵) compared to controls. The difference in θ_w and the number of USS were more pronounced when restricted to the coding region (p<1.31×10⁻²¹ and p = 0.006607, respectively). Further analysis of the region revealed non-synonymous variations were significantly higher in Complex I among the patients (p = 0.0053). Similar trends were retained when USS was considered only within complex I (frequency 0.49 vs 0.31 with p<0.0001 and mutation rate p-value <1.49×10⁻⁴³) and ND5 within its gene cluster (frequency 0.47 vs 0.23 with p<0.0001 and mutation rate p-value <4.42×10⁻⁴⁷). ND5 is involved in the proton pumping mechanism. Incidentally, glaucomatous trabecular meshwork cells have been reported to be more sensitive to inhibition of complex I activity. Thus mutations in ND5, expected to inhibit complex I activity, could lead to generation of oxidative stress and favor glaucomatous condition.     

Heavy metal ion interactions with deoxy nucleotides. X-ray crystal structures of orthorhombic and monoclinic forms of 2'-deoxy cytidine 5'-monophosphate complexed with Cd(II)

The crystal structures of two polymorphic complexes of Cd(II) with 2'-deoxy cytidine 5'-mono-phosphate (5'-dCMP) are reported and discussed The orthorhombic complex, [Cd(5'-dCMP) (H₂O)₂] (1), crystallizes in space group P2₁2₁2₁ with cell dimensions a = 8.422(2), b = 24.428(8), c = 7.292(2) Å, and Z = 4, the monoclinic complex, [Cd₂(5'-dCMP)₂(H₂O)₂]·3H₂O (2), crystallizes in space group C2 with a = 30.809(9), b = 5.369(2), c = 25.126(8) Å, β = 127.61(2)°, and Z = 4 Structure (1) has been solved by Patterson and Fourier methods, and structure (2) has been deduced from its isomorphous ribo-analog (J.K. Shiba and R. Bau, Inorg.Chem. 17, 3484 (1978)); and both have been refined by full-matrix least-squares methods to final R values of 0.052 for (1) and 0.103 for (2) using, respectively, 1273 and 2287 independent reflections. The orthorhombic complex (1) is three-dimensionally polymeric, with the Cd atom bound in a distorted octahedral arrangement to the cytosine base at N(3) and O(2) [Cd-N(3) 2.29(1) and Cd-O(2) 2.64(1) Å], to two phosphate oxygens of different nucleotide molecules, and to two water molecules, each phosphate group links two Cd atoms, thereby producing an infinite [-Cd-O-P-O-Cd-]_n spiraling column, which is additionally interconnected to neighboring columns via nucleotide bridges to give a layer-like polymeric structure. The monoclinic complex (2), which includes two crystallographically independent [Cd(5'-dCMP) (H2O)] units, is also three-dimensionally polymeric, with each Cd atom bound in a distorted pentagonal bipyramidal arrangement to N(3) and O(2) of the base [Cd-N(3) 2.35_av and Cd-O(2) 2.73_av Å], to four phosphate oxygens of three different nucleotide molecules, and to a water molecule, each phosphate group binds to three Cd atoms, thereby creating an infinite cross-linked spiral of sequences [-Cd-O-P-O-Cd-]_n and [-Cd-O-Cd-]_n that is further connected to adjacent spirals via nucleotide bridges to form large     

Purification and Characterization of Two Versiconal Hemiacetal Acetate Reductases Involved in Aflatoxin Biosynthesis

Two versiconal hemiacetal acetate (VHA) reductase activities (designated I and II), which catalyzed the reaction from VHA to versiconol acetate (VOAc) during aflatoxin biosynthesis, were purified to apparent homogeneity from the cytosol fraction of the mycelia of Aspergillus parasiticus mutant NIAH-26 through the following chromatography steps: first, fractionation with ammonium sulfate and then fractionation in succession with phenyl-Sepharose, DEAE-Sepharose, Sephacryl S-300, hydroxylapatite, and Matrex gel Green A chromatography. VHA reductase I and VHA reductase II were completely separated at the end of the DEAE-Sepharose step. The apparent molecular masses of reductase I and reductase II were estimated (by gel filtration) to be approximately 390 kDa; their denaturing molecular masses were 39- and 40-kDa, respectively (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Their pI values were 6.6 and 6.0, respectively (as determined by isoelectric focusing), and the optimal pH values were 8.0 and 9.0, respectively, although both enzymes exhibited a broad optimal pH range of between 7.5 and 9.0. The K_m values of reductase I and reductase II for VHA were 35.4 and 25.4 μM, respectively. On the other hand, in the cell-free experiments involving either VHA reductase fraction and high-performance liquid chromatography, both (2′S)- and (2′R)-VOAc enantiomers were formed from racemic VHA and more of the 2′R isomer than the 2′S isomer was produced, indicating that the VHA reductase fractions have very similar stereospecificities to the substrate.