Showing papers by "Christopher T. Walsh published in 2011"
TL;DR: The anatomy of the hip capsule is reviewed to provide a diagnosis-based consideration of capsular management during hip arthroscopy and the senior author's preferred techniques are presented.
Abstract: Advances in the ability to treat various soft-tissue and osseous pathologic conditions of the hip arthroscopically have been predicated on an improved exposure of the pathology of the central, peripheral, and peritrochanteric compartments. The management of the capsule is critical and must allow for an improved exposure without compromising stability and kinematics of the hip. Described approaches have included capsulectomy, limited capsulotomy, extensile capsulotomy, capsular plication, and capsular shift. The selected approach must consider various factors, including symptomatic complaints, underlying hyperlaxity, specific mechanical pathology, and surgical expertise. Universally using a single technique without consideration of the complex mechanical and anatomic factors unique to each patient may result in incomplete treatment of the pathoanatomy or iatrogenic instability. This article reviews the anatomy of the hip capsule and provide a diagnosis-based consideration of capsular management during hip arthroscopy. The senior author's preferred techniques are also presented.
170 citations
TL;DR: New genetic and biochemical insights are provided into the biosynthesis of this group of fungal alkaloids, including the tremorgens related to 2.
Abstract: Tremorgenic mycotoxins are a group of indole alkaloids which include the quinazoline-containing tryptoquivaline (2) that are capable of eliciting intermittent or sustained tremors in vertebrate animals. The biosynthesis of this group of bioactive compounds, which are characterized by an acetylated quinazoline ring connected to a 6−5−5 imidazoindolone ring system via a 5-membered spirolactone, has remained uncharacterized. Here, we report the identification of a gene cluster (tqa) from P. aethiopicum that is involved in the biosynthesis of tryptoquialanine (1), which is structurally similar to 2. The pathway has been confirmed to go through an intermediate common to the fumiquinazoline pathway, fumiquinazoline F, which originates from a fungal trimodular nonribosomal peptide synthetase (NRPS). By systematically inactivating every biosynthetic gene in the cluster, followed by isolation and characterization of the intermediates, we were able to establish the biosynthetic sequence of the pathway. An unusual o...
127 citations
TL;DR: This work has identified a minimum set of enzymes needed for generation of the pacidamycin scaffold from amino acid and nucleoside monomers, highlighting a freestanding thiolation domain (PacH) as a key carrier component in the peptidyl chain assembly as well as aFreestanding condensation (C)domain (PacI) catalyzing the release of the assembled peptide by a nucleosides moiety.
Abstract: Pacidamycins are a family of uridyl peptide antibiotics that inhibit the translocase MraY, an essential enzyme in bacterial cell wall biosynthesis that to date has not been clinically targeted. The pacidamycin structural skeleton contains a doubly inverted peptidyl chain with a β-peptide and a ureido linkage as well as a 3'-deoxyuridine nucleoside attached to DABA(3) of the peptidyl chain via an enamide linkage. Although the biosynthetic gene cluster for pacidamycins was identified recently, the assembly line of this group of peptidyl nucleoside antibiotics remained poorly understood because of the highly dissociated nature of the encoded nonribosomal peptide synthetase (NRPS) domains and modules. This work has identified a minimum set of enzymes needed for generation of the pacidamycin scaffold from amino acid and nucleoside monomers, highlighting a freestanding thiolation (T) domain (PacH) as a key carrier component in the peptidyl chain assembly as well as a freestanding condensation (C) domain (PacI) catalyzing the release of the assembled peptide by a nucleoside moiety. On the basis of the substrate promiscuity of this enzymatic assembly line, several pacidamycin analogues were produced using in vitro total biosynthesis.
70 citations
TL;DR: This study is the first to utilize an understanding of the molecular basis of scytonemin assembly to explore its synthesis and function within natural microbial communities.
68 citations
TL;DR: The gene cluster responsible for production of the elongation factor Tu (EF-Tu)-targeting 29-member thiazolyl peptide GE37468 from Streptomyces ATCC 55365 and its heterologous expression in the model host StrePTomyces lividans is reported, with an unusual β-methyl-δ-hydroxy-proline residue that may increase conformational rigidity of the macrocycle and impart reduced entropic costs of target binding.
Abstract: Thiazolyl peptides are bacterial secondary metabolites that potently inhibit protein synthesis in Gram-positive bacteria and malarial parasites. Recently, our laboratory and others reported that this class of trithiazolyl pyridine-containing natural products is derived from ribosomally synthesized preproteins that undergo a cascade of posttranslational modifications to produce architecturally complex macrocyclic scaffolds. Here, we report the gene cluster responsible for production of the elongation factor Tu (EF-Tu)-targeting 29-member thiazolyl peptide GE37468 from Streptomyces ATCC 55365 and its heterologous expression in the model host Streptomyces lividans. GE37468 harbors an unusual β-methyl-δ-hydroxy-proline residue that may increase conformational rigidity of the macrocycle and impart reduced entropic costs of target binding. Isotope feeding and gene knockout were employed in the engineered S. lividans strain to identify the P450 monooxygenase GetJ as the enzyme involved in posttranslational transformation of isoleucine 8 to β-methyl-δ-hydroxy-proline through a predicted tandem double hydroxylation/cyclization mechanism. Loss of Ile8 oxygenative cyclization or mutation of Ile8 to alanine via preprotein gene replacement resulted in a 4-fold and 2-fold drop in antibiotic activity, respectively. This report of genetic manipulation of a 29-member thiazolyl peptide sets the stage for further genetic examination of structure activity relationships in the EF-Tu targeting class of thiazolyl peptides.
68 citations
TL;DR: The characterization of PacB completes the assembly line reconstitution of pacidamycin pentapeptide antibiotic scaffolds, bridging the primary and secondary metabolic pathways by hijacking an aminoacyl-tRNA to the antibiotic biosynthetic pathway.
Abstract: Pacidamycins are a family of uridyl tetra/pentapeptide antibiotics with antipseudomonal activities through inhibition of the translocase MraY in bacterial cell wall assembly. The biosynthetic gene cluster for pacidamycins has recently been identified through genome mining of the producer Streptomyces coeruleorubidus, and the highly dissociated nonribosomal peptide assembly line for the uridyl tetrapeptide scaffold of pacidamycin has been characterized. In this work a hypothetical protein PacB, conserved in known uridyl peptide antibiotics gene clusters, has been characterized by both genetic deletion and enzymatic analysis of the purified protein. PacB catalyzes the transfer of the alanyl residue from alanyl-tRNA to the N terminus of the tetrapeptide intermediate yielding a pentapeptide on the thio-templated nonribosomal peptide synthetase (NRPS) assembly line protein PacH. PacB thus represents a new group of tRNA-dependent peptide bond-forming enzymes in secondary metabolite biosynthesis in addition to the recently identified cyclodipeptide synthases. The characterization of PacB completes the assembly line reconstitution of pacidamycin pentapeptide antibiotic scaffolds, bridging the primary and secondary metabolic pathways by hijacking an aminoacyl-tRNA to the antibiotic biosynthetic pathway.
60 citations
TL;DR: It is shown that the intramolecular disulfide bridge is constructed from the acyclic ene-dithiol at a late stage in the pathway by a thioredoxin oxidoreductase-like enzyme HlmI from the holomycin producer Streptomyces clavuligerus.
Abstract: Holomycin and related dithiolopyrrolone antibiotics display broad-spectrum antimicrobial activities and contain a unique 5,5-bicyclic ring structure with an N-acylated aminopyrrolone fused to a cyclic ene-disulfide. Here we show that the intramolecular disulfide bridge is constructed from the acyclic ene-dithiol at a late stage in the pathway by a thioredoxin oxidoreductase-like enzyme HlmI from the holomycin producer Streptomyces clavuligerus. Recombinant HlmI was purified from E. coli with bound flavin adenine dinucleotide (FAD) and converts reduced holomycin to holomycin utilizing O(2) as cosubstrate. As a dithiol oxidase, HlmI is functionally homologous to GliT and DepH, which perform a similar dithiol to disulfide oxidation in the biosynthesis of fungal natural product gliotoxin and epigenetic regulator compound FK228, respectively. Deletion of the hlmI gene in the wild type S. clavuligerus and in a holomycin-overproducing mutant resulted in decreased level of holomycin production and increased sensitivity toward holomycin, suggesting a self-protection role of HlmI in the holomycin biosynthetic pathway. HlmI belongs to a new clade of uncharacterized thioredoxin oxidoreductase-like enzymes, distinctive from the GliT-like enzymes and the DepH-like enzymes, and represents a third example of oxidoreductases that catalyzes disulfide formation in the biosynthesis of small molecules.
56 citations
TL;DR: Results indicate that adenylation domain promiscuity and relaxed downstream processing in the X. nematophila NRPS provide a conduit for xenematide diversification.
56 citations
TL;DR: Results demonstrate that a fourth halogenase, KthP, is active in the kutzneride biosynthetic pathway and suggest further processing of the (3S,5S)-5-chloropiperazate during subsequent incorporation into the kutschneride depsipeptide frameworks.
Abstract: Kutznerides 2 and 8 of the cyclic hexadepsipeptide family of antifungal natural products from the soil actinomycete Kutzneria sp. 744 contain two sets of chlorinated residues, a 6,7-dichlorohexahydropyrroloindole moiety derived from dichlorotryptophan and a 5-chloropiperazate moiety, as well as a methylcyclopropylglycine residue that may arise from isoleucine via a cryptic chlorination pathway. Previous studies identified KtzD, KtzQ, and KtzR as three halogenases in the kutzneride pathway but left no candidate for installing the C5 chlorine on piperazate. On the basis of analysis of the complete genome sequence of Kutzneria, we now identify a fourth halogenase in the pathway whose gene is separated from the defined kutzneride cluster by 12 open reading frames. KthP (kutzneride halogenase for piperazate) is a mononuclear nonheme iron halogenase that acts on the piperazyl ring tethered by a thioester linkage to the holo forms of thiolation domains. MS analysis of the protein-bound product confirmed chlorination of the piperazate framework from the (3S)- but not the (3R)-piperazyl-S-pantetheinyl thiolation proteins. After thioesterase-mediated release, nuclear magnetic resonance was used to assign the free imino acid as (3S,5S)-5-chloropiperazate, distinct from the 3S,5R stereoisomer reported in the mature kutznerides. These results demonstrate that a fourth halogenase, KthP, is active in the kutzneride biosynthetic pathway and suggest further processing of the (3S,5S)-5-chloropiperazate during subsequent incorporation into the kutzneride depsipeptide frameworks.
54 citations
TL;DR: This study shows that the fourth enzyme, the monocovalent flavoprotein Af12070, introduces a third layer of scaffold complexity by converting FQA to the spirohemiaminal FQC, presumably by catalyzing the formation of a transient imine within the pyrazinone ring (and therefore acting in an unprecedented manner as an FAD-dependent amide oxidase).
Abstract: The human pathogen Aspergillus fumigatus makes a series of fumiquinazoline (FQ) peptidyl alkaloids of increasing scaffold complexity using l-Trp, 2 equiv of l-Ala, and the non-proteinogenic amino acid anthranilate as building blocks. The FQ gene cluster encodes two non-ribosomal peptide synthetases (NRPS) and two flavoproteins. The trimodular NRPS Af12080 assembles FQF (the first level of complexity) while the next two enzymes, Af12060 and Af12050, act in tandem in an oxidative annulation sequence to couple alanine to the indole side chain of FQF to yield the imidazolindolone-containing FQA. In this study we show that the fourth enzyme, the monocovalent flavoprotein Af12070, introduces a third layer of scaffold complexity by converting FQA to the spirohemiaminal FQC, presumably by catalyzing the formation of a transient imine within the pyrazinone ring (and therefore acting in an unprecedented manner as an FAD-dependent amide oxidase). FQC subsequently converts nonenzymatically to the known cyclic aminal ...
53 citations
TL;DR: The current stage of research on biosynthetic pathways for peptidyl nucleoside antibiotics is discussed, primarily focusing on chemical logic and enzymatic machinery for uridine transformation and coupling to peptides.
Abstract: Peptidyl nucleoside antibiotics are a group of natural products targeting MraY, a bacterial translocase involved in the lipid-linked cycle in peptidoglycan biosynthesis. In this Perspective, we explore how Nature builds complex peptidyl nucleoside antibiotics scaffolds from simple nucleoside and amino acid building blocks. We discuss the current stage of research on biosynthetic pathways for peptidyl nucleoside antibiotics, primarily focusing on chemical logic and enzymatic machinery for uridine transformation and coupling to peptides. We further survey the nonribosomal biosynthetic paradigm for a subgroup of uridyl peptide antibiotics represented by pacidamycins, concluded by diversification opportunities for antibiotic optimization.
TL;DR: The terminal 50 kDa condensation domains of Af12050 and TqaB are solely responsible for the stereochemical control as shown both by making chimeric forms of these monomodular NRPS enzymes and by expression, purification, and assay of the excised C-domains.
Abstract: The fungal peptidyl alkaloids of the tryptoquialanine and fumiquinazoline families are nonribosomally assembled by annulation of the indole side chain of fumiquinazoline F (FQF) with an alaninyl or aminoisobutyryl unit by monomodular NRPS enzymes containing adenylation, thiolation, and condensation (A-T-C) domains. The Af12060 and Af12050 enzyme pair from Aspergillus fumigatus thereby converts FQF to FQA, while the homologous TqaH and TqaB enzyme pair from Penicillium aethiopicum makes the 2′-epi diastereomer of FQA, differing only in the stereochemistry of one of the C–N bonds formed in the annulation with l-Ala. To evaluate the basis for this stereochemical control, we have mixed and matched the flavoprotein oxygenases Af12060 and TqaH with the A-T-C modular enzymes Af12050 and TqaB to show that the NRPS enzymes control the stereochemical outcome. The terminal 50 kDa condensation domains of Af12050 and TqaB are solely responsible for the stereochemical control as shown both by making chimeric (e.g., A-T...
TL;DR: This work biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-T Tyr) from Phe, and sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.
Abstract: Phenylalanine hydroxylase (PheH) is an iron(II)-dependent enzyme that catalyzes the hydroxylation of aromatic amino acid l-phenylalanine (l-Phe) to l-tyrosine (l-Tyr). The enzymatic modification has been demonstrated to be highly regiospecific, forming proteinogenic para-Tyr (p-Tyr) exclusively. Here we biochemically characterized the first example of a phenylalanine 3-hydroxylase (Phe3H) that catalyzes the synthesis of meta-Tyr (m-Tyr) from Phe. Subsequent mutagenesis studies revealed that two residues in the active site of Phe3H (Cys187 and Thr202) contribute to C-3 rather than C-4 hydroxylation of the phenyl ring. This work sets the stage for the mechanistic and structural study of regiospecific control of the substrate hydroxylation by PheH.
TL;DR: It is shown here that L-H(2)Phe is a secreted metabolite in Photorhabdus luminescens cultures and a precursor of a recently described 2,5-dihydrostilbene, illustrating the versatile metabolic rerouting of prephenate from aromatic amino acid synthesis to antibiotic synthesis.
TL;DR: It is reported that the R,R-epoxyamide warhead is a more efficient covalent inactivator of glucosamine-6-phosphate synthase by 1 order of magnitude versus the eneamide, and this difference correlates with a >10-fold difference in antibiotic activity for the corresponding acyl-DAP dipeptides.
Abstract: The dapdiamides make up a family of antibiotics that have been presumed to be cleaved in the target cell to enzyme-inhibitory N-acyl-2,3-diaminopropionate (DAP) warheads containing two alternative electrophilic moieties. Our prior biosynthetic studies revealed that an eneamide warhead is made first and converted to an epoxyamide via a three-enzyme branch pathway. Here we provide a rationale for this logic. We report that the R,R-epoxyamide warhead is a more efficient covalent inactivator of glucosamine-6-phosphate synthase by 1 order of magnitude versus the eneamide, and this difference correlates with a >10-fold difference in antibiotic activity for the corresponding acyl-DAP dipeptides.
TL;DR: The enzymatic activation of 3,4-dehydrolysine and subsequent formation of the 12- Membered syringolin macrolactam were investigated and the SylD-TTE didomain was characterized and demonstrated to be the catalyst for formation of 12-membered macrocycles.