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The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >10(5)-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >10(5)-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3-18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A cocrystal structure of PTP1B bound with a nephrin pY(1193) peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities, and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed.

The multicopper oxidase phenoxazinone synthase (PHS) catalyzes the penultimate step in the biosynthesis of the antibiotic actinomycin D by Streptomyces antibioticus. PHS exists in two oligomeric forms: a dimeric form and a hexameric form, with older actinomycin-producing cultures containing predominately the hexameric form. The structure of hexameric PHS has been determined using X-ray diffraction to a resolution limit of 2.30 A and is found to contain several unexpected and distinctive features. The structure forms a hexameric ring that is centered on a pseudo 6-fold axis and has an outer diameter of 185 A with a large central cavity that has a diameter of 50 A. This hexameric structure is stabilized by a long loop connecting two domains; bound to this long loop is a fifth copper atom that is present as a type 2 copper. This copper atom is not present in any other multicopper oxidase, and its presence appears to stabilize the hexameric structure.

Simocyclinone D(8) consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A(1), the phenolic hydroxyl group of the aminocoumarin in simocyclinone is not glycosylated with a decorated noviosyl moiety that is the pharmacophore for targeting bacterial DNA gyrase. We have expressed the Streptomyces antibioticus simocyclinone ligase SimL, purified it from Escherichia coli, and established its ATP-dependent amide bond forming activity with a variety of polyenoic acids including retinoic acid and fumagillin. We have then used the last three enzymes from the novobiocin pathway, NovM, NovP, and NovN, to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetraenoate moiety, to evaluate antibacterial activity.

Eukaryotic phosphatidylinositol-specific phospholipase Cs (PI-PLCs) utilize calcium as a cofactor during catalysis, whereas prokaryotic PI-PLCs use a spatially conserved guanidinium group from Arg69. In this study, we aimed to construct a metal-dependent mutant of a bacterial PI-PLC and characterize the catalytic role of the metal ion, seeking an enhanced understanding of the functional differences between these two positively charged moieties. The following results indicate that a bona fide catalytic metal binding site was created by the single arginine-to-aspartate mutation at position 69: (1) The R69D mutant was activated by Ca(2+), and the activation was specific for R69D, not for other mutants at this position. (2) Titration of R69D with Ca(2+), monitored by (15)N-(1)H HSQC (heteronuclear single quantum coherence) NMR, showed that addition of Ca(2+) to R69D restores the environment of the catalytic site analogous to that attained by the WT enzyme. (3) Upon Ca(2+) activation, the thio effect of the S(P)-isomer of the phosphorothioate analogue (k(O)/k(Sp) = 4.4 x 10(5)) approached a value similar to that of the WT enzyme, suggesting a structural and functional similarity between the two positively charged moieties (Arg69 and Asp69-Ca(2+)). The R(P)-thio effect (k(O)/k(Rp) = 9.4) is smaller than that of the WT enzyme by a factor of 5. Consequently, R69D-Ca(2+) displays higher stereoselectivity (k(Rp)/k(Sp) = 47,000) than WT (k(Rp)/k(Sp) = 7600). (4) Results from additional mutagenesis analyses suggest that the Ca(2+) binding site is comprised of side chains from Asp33, Asp67, Asp69, and Glu117. Our studies provide new insight into the mechanism of metal-dependent and metal-independent PI-PLCs.

The interaction of a periplasmic nuclease, isolated from Streptomyces antibioticus, with several oligodeoxynucleotide substrates has been studied. Double-stranded oligonucleotides that contain sequences of four or more consecutive deoxyguanosine residues are preferentially hydrolyzed, with the strongest cutting site occurring at GGG decreases G. The enzyme does not hydrolyze these sequences in single-stranded DNA. However the sequence selectivity of the nuclease is far from absolute. Other sequences can also be cut, albeit more poorly, and differences in cutting rates are observed for runs of dG bases that differ in their flanking sequences. An oligonucleotide, thirty-six bases in length, that contains a central run of five dG bases has been used to evaluate the importance of the individual deoxyguanosines in recognition and cleavage. With this oligonucleotide cutting takes place at GG[symbol: see text]G decreases G[symbol: see text]G (decreases, most prominent cut; [symbol: see text], less prominent cuts). The use of dG base analogues revealed that two bases, one and two steps removed from the cleavage site in the 5' direction (*G*GG decreases), were of most importance in the determination of the nuclease DNA cleavage selectivity. Of these the inner starred dG was the most critical. The use of 5-methyldeoxycytidine also showed that the dC, base paired to this critical dG, influenced cleavage specificity. The overall pattern of results seen with the base analogues suggested that the nuclease interacted with both strands of the DNA and also contacted the nucleic acid in both the major and minor grooves. Gel retardation analysis together with footprinting experiments using hydroxyl radicals, dimethyl sulfate, and ethylnitrosourea indicated that the nuclease does not form a tight and specific complex with sequences containing dG runs, at least in the absence of the essential co-factor, Mg2+.