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Based on our first structural data of L-threonine dehydrogenase (TDH) of Pyrococcus horikoshii (PhTDH), we examined its catalytic mechanism. The structural analysis indicated that a catalytic zinc atom at the active centre of PhTDH is coordinated by four residues (Cys42, His67, Glu68 and Glu152) with low affinity. These residues are highly conserved in alcohol dehydrogenases (ADHs) and TDHs. Several PhTDH mutants were prepared with respect to Glu152 and other residues, relating to the proton relay system that is substantially a rate-limiting step in ADH. It was found that the E152D mutant showed 3-fold higher turnover rate and reduced affinities toward L-threonine and NAD(+), compared to wild-type PhTDH. The kinetic analysis of Glu152 mutants indicated that the carboxyl group of Glu152 is important for expressing the catalytic activity. The results obtained from pH dependency of kinetic parameters suggested that Glu152 to Asp substitution causes the enhancement of deprotonation of His47 or ionization of zinc-bound water and threonine in the enzyme-NAD(+) complex. Furthermore, it was predicted that the access of threonine substrate to the enzyme-NAD(+) complex induces a large conformational change in the active domain of PhTDH. From these results, we propose here that the proton relay system works as a catalytic mechanism of PhTDH.

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The cytochrome P450 enzyme-encoding genes rosC and rosD were cloned from the rosamicin biosynthetic gene cluster of Micromonospora rosaria IFO13697. The functions of RosC and RosD were demonstrated by gene disruption and complementation with M. rosaria and bioconversion of rosamicin biosynthetic intermediates with Escherichia coli expressing RosC and RosD. It is proposed that M. rosaria IFO13697 has two pathway branches that lead from the first desosaminyl rosamicin intermediate, 20-deoxo-20-dihydro-12,13-deepoxyrosamicin, to rosamicin. O xidation catalyzed by cytochrome P450 enzymes in postpolyketide synthase (post-PKS) modification of macrolide antibiotics contributes to structural diversification and modulates bioactivity. Rosamicin, which is a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697 (1), contains an epoxide and a formyl group at the C-12/13 and C-20 positions, respectively, and it is expected that two different types of P450s generate these functional groups (Fig. 1). Recently, we reported that the mycinosyl rosamicin derivatives were produced by genetic engineering of M. rosaria TPMA0001 (2, 3). Here, we cloned the cytochrome P450 enzyme-encoding genes rosC and rosD from M. rosaria IFO13697 and demonstrated the functions of RosC and RosD in the rosamicin biosynthetic pathway.Six complete protein-coding regions (orf1 to orf4, rosC, and rosD) and a partial protein-coding region (rosAI) were contained in the 9,036-bp DNA fragment in the cosmid pRS85, which was isolated using a PCR product amplified with degenerate primers as a DNA probe for colony hybridization. The primers were designed from two conserved regions of deduced amino acid sequences of P450s implicated in formylation of 16-member macrolides (4-8). The complete nucleotide sequence of the rosamicin biosynthetic gene cluster in Micromonospora carbonacea subsp. aurantiaca NRRL2997 was determined by Farnet et al. (8). The deduced amino acid sequences of RosC and RosD were most similar to P450s encoded in ORF3 and ORF4 of M. carbonacea subsp. aurantiaca NRRL 2997 (87% and 83% identity, respectively) (see Fig. S1 in the supplemental material). In BLAST searches, RosC and RosD were similar to TylI (71% identity) in tylosin biosynthesis and OleP (48% identity) in oleandomycin biosynthesis, respectively (4, 9).To obtain the rosC and rosD disruption mutants of M. rosaria IFO13697, disruption plasmids pRS511 and pRS514 were constructed using a PCR-targeting method (10). These disruption plasmids were introduced into M. rosaria IFO13697 by conjugation using our previous procedure (2). The resulting disruption mutants, TPMA0050 and TPMA0055, did not produce rosamicin when the strains were cultured in 172F medium. However, an unknown compound, RS-B, accumulated in the TPMA0050 culture broth, and unknown peaks RS-C, RS-D, and RS-E were detected in ethyl acetate extract of the TPMA0055 culture broth by high-performance liquid chromatography (HPLC) analysis (Fig. 2; see Fig. S2 in the supplemental material). Furthermore, when ros...

The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii, was cloned and overexpressed in Escherichia coli. The recombinant enzyme was determined to be thermostable aminoacylase (PhoACY), forming a homotetramer. Purified PhoACY showed the ability to release amino acid molecules from the substrates N‐acetyl‐l‐Met, N‐acetyl‐l‐Gln and N‐acetyl‐l‐Leu, but had a lower hydrolytic activity towards N‐acetyl‐l‐Phe. The kinetic parameters Km and kcat were determined to be 24.6 mm and 370 s−1, respectively, for N‐acetyl‐l‐Met at 90 °C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn2+, Mn2+ and Ni2+), and Mn2+ addition caused an alteration in substrate specificity. Site‐directed mutagenesis analysis and structural modeling of PhoACY, based on Arabidopsis thaliana indole‐3‐acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, His106, Glu139, Glu140 and His164 were related to the metal‐binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.