Ayumi Yoshizumi

Production of a doubly chiral compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, by two-step enzymatic asymmetric reduction.

ABSTRACT A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD+, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.

Old Yellow Enzyme from Candida macedoniensis catalyzes the stereospecific reduction of the C=C bond of ketoisophorone.

Microorganisms were screened for ones that reduced 3,5,5-trimethyl-2-cyclohexene-1,4-dione (ketoisophorone; KIP), and several strains were found to produce (6R)-2,2,6-trimethylcyclohexane-1,4-dione (levodione). The enzyme catalyzing the reduction of the C=C bond of KIP to yield (6R)-levodione was isolated from Candida macedoniensis AKU4588. The results of primary structural analysis and its enzymatic properties suggested that the enzyme might be an Old Yellow Enzyme family protein.

Self-association of Streptococcus pyogenes collagen-like constructs into higher order structures

A number of bacterial collagen‐like proteins with Gly as every third residue and a high Pro content have been observed to form stable triple‐helical structures despite the absence of hydroxyproline (Hyp). Here, the high yield cold‐shock expression system is used to obtain purified recombinant collagen‐like protein (V‐CL) from Streptococcus pyogenes containing an N‐terminal globular domain V followed by the collagen triple‐helix domain CL and the modified construct with two tandem collagen domains V‐CL‐CL. Both constructs and their isolated collagenous domains form stable triple‐helices characterized by very sharp thermal transitions at 35–37°C and by high values of calorimetric enthalpy. Procedures for the formation of collagen SLS crystallites lead to parallel arrays of in register V‐CL‐CL molecules, as well as centrosymmetric arrays of dimers joined at their globular domains. At neutral pH and high concentrations, the bacterial constructs all show a tendency towards aggregation. The isolated collagen domains, CL and CL‐CL, form units of diameter 4–5 nm which bundle together and twist to make larger fibrillar structures. Thus, although this S. pyogenes collagen‐like protein is a cell surface protein with no indication of participation in higher order structure, the triple‐helix domain has the potential of forming fibrillar structures even in the absence of hydroxyproline. The formation of fibrils suggests bacterial collagen proteins may be useful for biomaterials and tissue engineering applications.

A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial.

A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not elicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37 degrees C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering.

Occurrence of multiple ethyl 4-chloro-3-oxobutanoate-reducing enzymes in Candida magnoliae

Multiple ethyl 4-chloro-3-oxobutanoate (COBE)-reducing enzymes were isolated from a cell-free extract of Candida magnoliae. A NADPH-dependent COBE-reducing enzyme, distinct from the carbonyl reductase and aldehyde reductase previously reported, was purified to homogeneity using five steps, including polyethylene glycol treatment. The relative molecular mass of the enzyme was estimated to be 86,000 on high performance gel-permeation chromatography and 29,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme catalyzed the reduction of COBE to the corresponding (S)-alcohol with a 51% enantiomeric excess. The substrate specificity of the enzyme was different from those of the other COBE-reducing enzymes of the same strain. The partial amino acid sequences of the enzyme showed that it belongs to the short chain alcohol dehydrogenase/reductase (SDR) family. This is the first report of multiple COBE-reducing enzymes with various stereoselectivities occurring in the same strain but belonging to different (super)families.

Designed coiled coils promote folding of a recombinant bacterial collagen.

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

Noncollagenous region of the streptococcal collagen-like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated Vsp, adjacent to its triple‐helix domain. The Vsp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant Vsp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of Tm = 45°C. Examination of a new construct shows that the Vsp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the Vsp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.

Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation.

The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)n sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.

Cloning and Overexpression of the Exiguobacterium sp. F42 Gene Encoding a New Short Chain Dehydrogenase, Which Catalyzes the Stereoselective Reduction of Ethyl 3-Oxo-3-(2-thienyl)propanoate to Ethyl (S)-3-Hydroxy-3-(2-thienyl)propanoate

Exiguobacterium sp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coli by simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES.