Replacement of Tyrosines by Unnatural Amino Acid Aminophenylalanine Leads to Metal-Mediated Aniline Free Radical Formation in a Copper Amine Oxidase.

Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to aldehyde, ammonia, and hydrogen peroxide as products and are widely distributed in bacteria, plants, and eukaryotes. These enzymes initiate the single turnover, post-translational conversion of an active site tyrosine to the redox cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ), subsequently employing TPQ to catalyze steady-state amine oxidation. The mechanisms of TPQ biogenesis and steady-state amine oxidation have been studied extensively, with consensus mechanisms proposed for both reactions. One unresolved issue has been whether the Cu2+ center must undergo formal reduction to Cu1+ in the course of the reaction. Herein, we investigate the properties of the active site of a yeast (Hansenula polymorpha) amine oxidase (HPAO) that has undergone site-specific insertion of a para-aminophenylalanine (pAF) into the position of either the precursor tyrosine to TPQ (Y405) or the two strictly conserved neighboring tyrosines (Y305 and Y407). While our original intention was to interrogate cofactor biogenesis using a precursor unnatural amino acid (UAA) of altered redox potential and pKa, we instead observe an unanticipated reaction assigned to an intramolecular electron transfer from pAF to the active site copper ion. We establish the generality of the observed active site chemistry using exogenously added, aniline-containing substrates under conditions that prevent side chain amine oxidation. The results support previous proposals that the activation of the TPQ precursor occurs in the absence of a formal valence change at the active site copper site. The described reaction of pAFs with the active site redox Cu2+ center of HPAO provides a prototype for either the engineering of the enzymatic oxidation of exogenous anilines or the insertion of site-specific free radical probes within proteins.

Discovery of Hydroxylase Activity for PqqB Provides a Missing Link in the Pyrroloquinoline Quinone Biosynthetic Pathway.

Understanding the biosynthesis of cofactors is fundamental to the life sciences, yet to date a few important pathways remain unresolved. One example is the redox cofactor pyrroloquinoline quinone (PQQ), which is critical for C1 metabolism in many microorganisms, a disproportionate number of which are opportunistic human pathogens. While the initial and final steps of PQQ biosynthesis, involving PqqD/E and PqqC, have been elucidated, the precise nature and order of the remaining transformations in the pathway are unknown. Here we show evidence that the remaining essential biosynthetic enzyme PqqB is an iron-dependent hydroxylase catalyzing oxygen-insertion reactions that are proposed to produce the quinone moiety of the mature PQQ cofactor. The demonstrated reactions of PqqB are unprecedented within the metallo ß-lactamase protein family and expand the catalytic repertoire of nonheme iron hydroxylases. These new findings also generate a nearly complete description of the PQQ biosynthetic pathway.

Chemo-enzymatic synthesis of the exocyclic olefin isomer of thymidine monophosphate.

Exocyclic olefin variants of thymidylate (dTMP) recently have been proposed as reaction intermediates for the thymidyl biosynthesis enzymes found in many pathogenic organisms, yet synthetic reports on these materials are lacking. Here we report two strategies to prepare the exocyclic olefin isomer of dTMP, which is a putative reaction intermediate in pathogenic thymidylate biosynthesis and a novel nucleotide analog. Our most effective strategy involves preserving the existing glyosidic bond of thymidine and manipulating the base to generate the exocyclic methylene moiety. We also report a successful enzymatic deoxyribosylation of a non-aromatic nucleobase isomer of thymine, which provides an additional strategy to access nucleotide analogs with disrupted ring conjugation or with reduced heterocyclic bases. The strategies reported here are straightforward and extendable towards the synthesis of various pyrimidine nucleotide analogs, which could lead to compounds of value in studies of enzyme reaction mechanisms or serve as templates for rational drug design.

Folate binding site of flavin-dependent thymidylate synthase.

The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH(2)H(4)folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.

Trapping of an intermediate in the reaction catalyzed by flavin-dependent thymidylate synthase.

Thymidylate is a DNA nucleotide that is essential to all organisms and is synthesized by the enzyme thymidylate synthase (TSase). Several human pathogens rely on an alternative flavin-dependent thymidylate synthase (FDTS), which differs from the human TSase both in structure and molecular mechanism. It has recently been shown that FDTS catalysis does not rely on an enzymatic nucleophile and that the proposed reaction intermediates are not covalently bound to the enzyme during catalysis, an important distinction from the human TSase. Here we report the chemical trapping, isolation, and identification of a derivative of such an intermediate in the FDTS-catalyzed reaction. The chemically modified reaction intermediate is consistent with currently proposed FDTS mechanisms that do not involve an enzymatic nucleophile, and it has never been observed during any other TSase reaction. These findings establish the timing of the methylene transfer during FDTS catalysis. The presented methodology provides an important experimental tool for further studies of FDTS, which may assist efforts directed toward the rational design of inhibitors as leads for future antibiotics.

Mechanisms and inhibition of uracil methylating enzymes.

Uracil methylation is essential for survival of organisms and passage of information from generation to generation with high fidelity. Two alternative uridyl methylation enzymes, flavin-dependent thymidylate synthase and folate/FAD-dependent RNA methyltransferase, have joined the long-known classical enzymes, thymidylate synthase and SAM-dependent RNA methyltransferase. These alternative enzymes differ significantly from their classical counterparts in structure, cofactor requirements and chemical mechanism. This review covers the available structural and mechanistic knowledge of the classical and alternative enzymes in biological uracil methylation, and offers a possibility of using inhibitors specifically aiming at microbial thymidylate production as antimicrobial drugs.

Flavin-dependent thymidylate synthase: a novel pathway towards thymine.

For several decades only one chemical pathway was known for the de novo biosynthesis of the essential DNA nucleotide, thymidylate. This reaction catalyzed by thyA or TYMS encoded thymidylate synthases is the last committed step in the biosynthesis of thymidylate and proceeds via the reductive methylation of uridylate. However, many microorganisms have recently been shown to produce a novel, flavin-dependent thymidylate synthase encoded by the thyX gene. Preliminary structural and mechanistic studies have shown substantial differences between these deoxyuridylate-methylating enzymes. Recently, both the chemical and kinetic mechanisms of FDTS have provided further insight into the distinctions between thyA and thyX encoded thymidylate synthases. Since FDTSs are found in several severe human pathogens their unusual mechanism offers a promising future for the development of antibiotic and antiviral drugs with little effect on human thymidylate biosynthesis.

Oxidase activity of a flavin-dependent thymidylate synthase.

Flavin-dependent thymidylate synthases (FDTS) catalyze the production of dTMP from dUMP and N(5),N(10)-methylene-5,6,7,8-tetrahydrofolate (CH(2)H(4)folate). In contrast to human and other classical thymidylate synthases, the activity of FDTS depends on a FAD coenzyme, and its catalytic mechanism is very different. Several human pathogens rely on this recently discovered enzyme, making it an attractive target for novel antibiotics. Like many other flavoenzymes, FDTS can function as an oxidase, which catalyzes the reduction of O(2) to H(2)O(2), using reduced NADPH or other reducing agents. In this study, we exploit the oxidase activity of FDTS from Thermatoga maritima to probe the binding and release features of the substrates and products during its synthase activity. Results from steady-state and single-turnover experiments suggest a sequential kinetic mechanism of substrate binding during FDTS oxidase activity. CH(2)H(4)folate competitively inhibits the oxidase activity, which indicates that CH(2)H(4)folate and O(2) compete for the same reduced and dUMP-activated enzymatic complex (FDTS-FADH(2)-NADP(+)-dUMP). These studies imply that the binding of CH(2)H(4)folate precedes NADP(+) release during FDTS activity. The inhibition constant of CH(2)H(4)folate towards the oxidase activity was determined to be rather small (2 microm), which indicates a tight binding of CH(2)H(4)folate to the FDTS-FADH(2)-NADP(+)-dUMP complex.

An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene.

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase to catalyse the methylation of the uracil moiety of 2'-deoxyuridine-5'-monophosphate. All known thymidylate synthases rely on an active site residue of the enzyme to activate 2'-deoxyuridine-5'-monophosphate. This functionality has been demonstrated for classical thymidylate synthases, including human thymidylate synthase, and is instrumental in mechanism-based inhibition of these enzymes. Here we report an example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (that is, a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2'-deoxythymidine-5'-monophosphate. These observations indicate a very different chemical cascade than that of classical thymidylate synthases or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Because several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

The relationships between oxidase and synthase activities of flavin dependent thymidylate synthase (FDTS).

New findings lead to a revised understanding of the substrates' binding order, the role of the substrate as an activator, and the observed lag phase in the FDTS catalyzed reaction.