Starch-degrading, amylolytic enzymes are widely distributed among microbes. Several activities are required to hydrolyze starch to its glucose units. These enzymes include alpha-amylase, beta-amylase, glucoamylase, alpha-glucosidase, pullulan-degrading enzymes, exoacting enzymes yielding alpha-type endproducts, and cyclodextrin glycosyltransferase. Properties of these enzymes vary and are somewhat linked to the environmental circumstances of the producing organisms. Features of the enzymes, their action patterns, physicochemical properties, occurrence, genetics, and results obtained from cloning of the genes are described. Among all the amylolytic enzymes, the genetics of alpha-amylase in Bacillus subtilis are best known. Alpha-Amylase production in B. subtilis is regulated by several genetic elements, many of which have synergistic effects. Genes encoding enzymes from all the amylolytic enzyme groups dealt with here have been cloned, and the sequences have been found to contain some highly conserved regions thought to be essential for their action and/or structure. Glucoamylase appears usually in several forms, which seem to be the results of a variety of mechanisms, including heterogeneous glycosylation, limited proteolysis, multiple modes of mRNA splicing, and the presence of several structural genes.

SnoaL belongs to a family of small polyketide cyclases, which catalyse ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. Several of these antibiotics are among the most used anti-cancer drugs currently in use. The crystal structure of SnoaL, involved in nogalamycin biosynthesis, with a bound product, has been determined to 1.35 Å resolution. The fold of the subunit can be described as a distorted a þ b barrel, and the ligand is bound in the hydrophobic interior of the barrel. The 3D structure and site-directed mutagenesis experiments reveal that the mechanism of the intramolecular aldol condensation catalysed by SnoaL is different from that of the classical aldolases, which employ covalent Schiff base formation or a metal ion cofactor. The invariant residue Asp121 acts as an acid/base catalyst during the reaction. Stabilisation of the enol(ate) intermediate is mainly achieved by the delocalisation of the electron pair over the extended p system of the substrate. These polyketide cyclases thus form of family of enzymes with a unique catalytic strategy for aldol condensation.

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the ␥-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.Soil actinomycetes, especially those that belong to the genus Streptomyces, have been the focus of intensive research for the past several decades. The interest in Streptomyces arose from the finding that this group of bacteria seems to have the ability to produce a large variety of different bioactive compounds that have a wide spectrum of activity. From the 1950s to the mid-1970s numerous new bioactive molecules were discovered through large screening programs, and these molecules subsequently found their way into various clinical uses ranging from control of infections to cancer treatment (21).In more recent years, modern high-throughput screening methods have exponentially increased the number of strains screened annually, but the number of novel compounds discovered has not increased in the same proportion. One of the many reasons, presumably the most important one, for this problem is that old molecules (and strains) are being rediscovered with the screening procedures that are in use today (21).In a previous paper (18) Metsä-Ketelä et al. reported a method that could be used for preliminary classification of strains on the basis of their genetic abilities to produce various compounds belonging to the aromatic polyketide group. This method is based on PCR amplification of a gene fragment that is essential in the biosynthesis pathway of aromatic polyketides and on analysis of the amplified regions by phylogenetic methods. The degenerate primers designed for this purpose amplify a portion of a ketosynthase gene (KS ␣ ), which in collaboration with KS ␤ and an acyl carrier protein condenses small carboxylic acids in a stepwise manner to form a long polyketide chain that is subsequently folded into a range of different aromatic compounds by various ketoreductases, cyclases, and aromatases. Later, the molecule formed is of...

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One of the final steps in the biosynthesis of the widely used anti-tumor drug daunorubicin in Streptomyces peucetius is the methylation of the 4-hydroxyl group of the tetracyclic ring system. This reaction is catalyzed by the S-adenosyl-L-methionine-dependent carminomycin 4-Omethyltransferase DnrK. The crystal structure of the ternary complex of this enzyme with the bound products S-adenosyl-L-homocysteine and 4-methoxy-⑀-rhodomycin T has been determined to a 2.35-Å resolution. DnrK is a homodimer, and the subunit displays the typical fold of small molecule O-methyltransferases. The structure provides insights into the recognition of the anthracycline substrate and also suggests conformational changes as part of the catalytic cycle of the enzyme. The position and orientation of the bound ligands are consistent with an S N 2 mechanism of methyl transfer. Mutagenesis experiments on a putative catalytic base confirm that DnrK most likely acts as an entropic enzyme in that rate enhancement is mainly due to orientational and proximity effects. This contrasts the mechanism of DnrK with that of other O-methyltransferases where acid/base catalysis has been demonstrated to be an essential contribution to rate enhancement.Daunorubicin and doxorubicin are aromatic polyketide antibiotics that exhibit high cytotoxicity and are widely applied in the chemotherapy of a variety of cancers (1, 2). These and related anthracyclines consist of a cyclic polyketide backbone, 7,8,9,10-tetrahydrotetracene-5,12-quinone, glycosylated at position C7 or C10 (Fig. 1). Diversity is generated by variations in the modification of the aglycone moiety and the composition of the attached carbohydrate. Biosynthesis of daunorubicin/doxorubicin starts with the formation of the polyketide backbone catalyzed by a class II polyketide synthase with subsequent cyclization of the polyketide chain (3). These steps lead to the formation of aklavinone, a common intermediate in the synthesis of most anthracyclines. This aglycone is then further modified through a series of steps, i.e. hydroxylation, glycosylation, methylester hydrolysis, decarboxylation, methylation, and, in

The yabJ gene in Bacillus subtilis is required for adenine-mediated repression of purine biosynthetic genes in vivo and codes for an acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is unknown. YabJ is a member of a large, widely distributed family of proteins of unknown biochemical function. The 1.7-Å crystal structure of YabJ reveals a trimeric organization with extensive buried hydrophobic surface and an internal water-filled cavity. The most important finding in the structure is a deep, narrow cleft between subunits lined with nine side chains that are invariant among the 25 most similar homologs. This conserved site is proposed to be a binding or catalytic site for a ligand or substrate that is common to YabJ and other members of the YER057c͞YjgF͞ UK114 family of proteins.

Aclacinomycin 10-hydroxylase is a methyltransferase homologue that catalyzes a S-adenosyl-L-methionine (AdoMet)-dependent hydroxylation of the C-10 carbon atom of 15-demethoxy-⑀-rhodomycin, a step in the biosynthesis of the polyketide antibiotic ␤-rhodomycin. SAdenosyl-L-homocysteine is an inhibitor of the enzyme, whereas the AdoMet analogue sinefungin can act as cofactor, indicating that a positive charge is required for catalysis.18 O 2 experiments show that the hydroxyl group is derived from molecular oxygen.

Tailoring steps in aromatic polyketide antibiotic biosynthesis are an important source of structural diversity and, consequently, an intriguing focal point for enzymological studies. PgaE and PgaM from Streptomyces sp. PGA64 are representatives of flavoenzymes catalyzing early post-PKS reactions in angucycline biosynthesis. This in vitro study illustrates that the chemoenzymatic conversion of UWM6 into the metabolite, gaudimycin C, requires multiple closely coupled reactions to prevent intermediate degradation. The NMR structure of gaudimycin C confirms that the reaction cascade involves C12- and C12b-hydroxylation, C2,3-dehydration, and stereospecific ketoreduction at C6. Enzymatic 18O incorporation studies verify that the oxygens at C12 and C12b derive from O2 and H2O, respectively. The results indicate that PgaM deviates mechanistically from flavoprotein monooxygenases, and suggest an alternative catalytic mechanism involving a quinone methide intermediate.