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Research has been conducted on the ketoreductases. The investigation of the beta-ketoacyl reductase stereocontrol has been carried out and the results indicate that the stereochemical course of reduction can be monitored via specific substrate analogues and a general substrate analogue modeling substrate for the ketoreductase fatty acid synthase activity.

The His244 residue of adenosylcobalamin-dependent methylmalonyl-CoA mutase appears to be involved in shielding the active site from water to protect the reactive radical intermediates formed during catalysis. The active site is deeply buried inside the enzyme and an His244 mutant created a cavity in the active site containing a water molecule.

There may be at least three specific roles for methylmalonyl-CoA in the AdoCbl-dependent skeletal rearrangement catalyzed by methylmalonyl-CoA mutase. There is little information about the involvement of active-site amino acids in catalysis of the radical rearrangement. Only a few polar active-site amino acids were found to be in direct contact with the substrate, particularly histidine 244, tyrosine 89 and arginine 207.

Complex polyketides that provide innumerable, significant biological properties are bio-synthesized by polyketide synthases (PKS). PKS-derived compounds are structurally diverse. Keen interest has been expressed for combining the functions of different PKS-derived compounds through genetic engineering to produce a vast amount of innovative compounds. A variety of strategies have been suggested including the use of the beta-ketosynthase (KS) domain of the module 1 found in PKSs using a bimodular derivative of the PKS called 6-deoxyerythronolide B synthase (DEBS).

Modular type I polyketide synthases (PKSs), including 6-deoxyerythronolide B synthase (DEBS), are responsible for the synthesis of macrolide antibiotic erythromycin. Macrolides were noted by Celmer to have the same configuration at all comparable sterocenters. In vitro studies with DEBS 1-TE, the bimodular derivative of DEBS from Saccharopolyspora erythraea, aimed to elucidate the molecular basis of Celmer's rules. It is shown that condensation of (2S)-methylmalonyl-CoA in module 2 proceeds without cleavage of the C-H bond next tot he methyl group, although the process involves loss of hydrogen in module 1.

The effects of tritium loss from 5'-[3H]adenosylcobalamin during the isomerization of methylmalonyl-CoA and succinyl-CoA and of its subsequent appearance in the substrate and reaction product were investigated by measuring the kinetics of the reaction. The primary kinetic tritium isotope of 4.9 on enzyme-catalyzed hydrogen transfer was found to be lower than the normal range, suggesting that hydrogen transfer is not a rate-limiting step in the reaction.

Aminoglycosides are essential antibiotics used to treat severe infections caused mainly by Gram-negative bacteria. Gentamicin is an aminoglycoside and, despite its toxicity, is clinically used to treat several pulmonary and urinary infections. The commercial form of gentamicin is a mixture of five compounds with minor differences in the methylation of one of their aminosugars. In the case of two compounds, gentamicin C2 and C2a, the only difference is the stereochemistry of the methyl group attached to C-6'. GenB2 is the enzyme responsible for this epimerization and is one of the four PLP-dependent enzymes encoded by the gentamicin biosynthetic gene cluster. Herein, we have determined the structure of GenB2 in its holo form in complex with PMP and also in the ternary complex with gentamicin X2 and G418, two substrate analogues. Based on the structural analysis, we were able to identify the structural basis for the catalytic mechanism of this enzyme, which was also studied by site-directed mutagenesis. Unprecedently, GenB2 is a PLP-dependent enzyme from fold I, which is able to catalyze an epimerization but with a mechanism distinct from that of fold III PLP-dependent epimerases using a cysteine residue near the N-terminus. The substitution of this cysteine residue for serine or alanine completely abolished the epimerase function of the enzyme, confirming its involvement. This study not only contributes to the understanding of the enzymology of gentamicin biosynthesis but also provides valuable details for exploring the enzymatic production of new aminoglycoside derivatives.

Neoantimycins (NATs) are members of antimycin-types of depsipeptides with outstanding anticancer activities. We isolated NAT-A (1) and -F (2) from the fermentation extract of Streptomyces conglobatus. The NAT biosynthetic gene cluster ( nat BGC) was identified by genome sequencing and bioinformatics analysis. nat BGC includes two nonribosomal peptide synthetase (NRPS) and one polyketide synthase (PKS) gene, and a gene cassette (10 genes), of which the encoded enzymes share high homology to the ones responsible for 3-formamidosalicylate (3-FAS) biosynthesis in the antimycin biosynthetic pathway. Heterologous expression of the partial nat BGC without the 3-FAS gene cassette in the antimycin producer, Streptomyces albus J1074, results in the production of 1 and 2, suggesting that the nat BGC indeed directs NATs biosynthesis. Targeted in-frame deletion of the reductase gene ( natE) abolished the production of 1 and 2 but accumulated two NAT derivatives, the known NAT-H (3) and a new NAT-I (4). Biochemical verification demonstrated that the recombinant NatE indeed catalyzes an NADPH-dependent reaction of 3 or 4 to 1 or 2, respectively. Compound 3 presented significantly stronger activities against eight cancer cell lines than the ones using cisplatin, the clinical chemotherapy medicine. In particular, 3 displayed 559- and 57-fold higher activity toward human melanoma and cervix epidermoid carcinoma cells, respectively, compared with cisplatin. The new derivative, 4, was 1.5- to 10.9-fold more active than cisplatin toward five cancer cell lines. The evaluation of NATs biosynthesis depicted here will pave the way to generate new NAT derivatives through rational pathway engineering.

Gentamicins are heavily methylated, clinically valuable pseudotrisaccharide antibiotics produced by Micromonospora echinospora. GenN has been characterized as an S-adenosyl-l-methionine-dependent methyltransferase with low sequence similarity to other enzymes. It is responsible for the 3″-N-methylation of 3″-dehydro-3″-amino-gentamicin A2, an essential modification of ring III in the biosynthetic pathway to the gentamicin C complex. Purified recombinant GenN also efficiently catalyzes 3″-N-methylation of related aminoglycosides kanamycin B and tobramycin, which both contain an additional hydroxymethyl group at the C5″ position in ring III. We have obtained eight cocrystal structures of GenN, at a resolution of 2.2 Å or better, including the binary complex of GenN and S-adenosyl-l-homocysteine (SAH) and the ternary complexes of GenN, SAH, and several aminoglycosides. The GenN structure reveals several features not observed in any other N-methyltransferase that fit it for its role in gentamicin biosynthesis. These include a novel N-terminal domain that might be involved in protein:protein interaction with upstream enzymes of the gentamicin X2 biosynthesis and two long loops that are involved in aminoglycoside substrate recognition. In addition, the analysis of structures of GenN in complex with different ligands, supported by the results of active site mutagenesis, has allowed us to propose a catalytic mechanism and has revealed the structural basis for the surprising ability of native GenN to act on these alternative substrates.

Modular type I polyketide synthases (PKSs) such as the 6-deoxyerythronolide B synthase (DEBS) or the rapamycin synthase (RAPS) biosynthesize their polyketide products in a fashion similar to fatty acid biosynthesis. Each module of these enzymes consists of multiple catalytic domains. The constituent enoylreductase (ER) domain of a given module stereospecifically reduces an enzyme-bound 2-enoyl intermediate. In a recombinant model PKS containing an ER domain derived from module 13 of RAPS, we have previously used site-specific mutagenesis to identify a key active site residue that influences the stereochemistry of enoylreduction. In this study we have identified further residues involved in stereospecificity. We show here that several other residues, previously considered as catalytically important in the medium-chain dehydrogenase/reductase family of enzymes to which PKS ERs belong, are not essential for enoylreduction in polyketide biosynthesis. However, our results suggest that a lysine residue, also modeled to lie at the active site, might serve as a proton donor to the C-2 position during enoylreduction, as previously proposed for an analogously placed lysine in mammalian fatty acid synthase. These findings further highlight the close mechanistic link between fatty acid and polyketide synthases and provide useful guidance for future biosynthetic engineering of complex polyketide biosynthesis.