HighlightsThe bacterial metabolic pathway of trans-3-hydroxy-l-proline (T3LHyp) has been identified.Azospirillum brasilense can grow on T3LHyp as a sole carbon source.The T3LHyp pathway comprises T3LHyp dehydratase and Δ1-pyrroline-2-carboxylate reductase.This pathway is similar to one found in mammals.

Post-translational hydroxylation of the L-proline residue mainly occurs in collagen; therefore, the L-hydroxyprolines (L-Hyp) synthesized, including trans-4-hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp), are important markers for directly measuring the content of collagen in several biological samples. The most frequently used method to estimate the content of L-Hyp is high-performance liquid chromatography (HPLC), which is inconvenient. In the present study, we attempted to estimate the content of L-Hyp using coupling systems with metabolic enzymes of the T4LHyp (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and T3LHyp pathways (T3LHyp dehydratase (T3LHypD) and Δ(1)-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. We constructed a functional expression system of recombinant HypDH with a heterooligomeric structure in Escherichia coli cells. Enzymological characterization revealed that the β-subunit acted as a catalytic subunit, and also that assembly with other subunit(s) improved the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of T4LHyp and T3LHyp were successfully estimated within the ranges of 0.004-1mM and 0.05-1mM, respectively, and were consistent with those determined by HPLC. This enzymatic method was used to measure the content of T4LHyp in the acid-hydrolysate of collagen, and blood plasma.

Proline racemase (ProR) is a member of the pyridoxal 5’-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR.　A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.

α-Functionalized acrylamides have not been considered as an effective monomer design due to their poor polymerizability, although the analogues, α-functionalized acrylates, are attractive monomers of which polymers exhibit characteristic properties. In this article, we report the first example of radical polymerization of α-functionalized N,N-disubstituted acrylamide affording thermo-responsive hydrophilic polymers. N,N-dimethyl-α-(hydroxymethyl)acrylamide (DMαHAA) was (co)polymerized with N,N-diethylacrylamide (DEAA). Although the homopolymerization did not afford a polymeric product, the copolymerizations with various feed ratios yielded a series of the copolymers containing 0%-65% of DMαHAA units. The obtained copolymers exhibited a lower critical solution temperature (LCST) in water; the cloud points (T c s) were linearly elevated as the contents of DMαHAA units from 32 to 64 • C, indicating that DMαHAA functioned as a more hydrophilic monomer than DEAA. The linear relationship between T c and DMαHAA content suggests that the homopolymer, poly(DMαHAA), should have T c at ca. 80 • C, although it is not available by direct radical homopolymerization.

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