Biology subjects

McKinnie, S. M. K.

Publications and source records attributed to McKinnie, S. M. K..

10 recordsLinked to original sources

bioRxiv2025

Integrated metabolomic and genomic insights into amino acid incorporation within the hybrid polyketide-alkaloid antibiotic TLN-05220

Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry. Micromonospora echinospora subsp. challisensis NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens including vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus. The pentangular polyphenol core of TLN-05220 is decorated with a piperazinone moiety, yet the enzymes responsible for the construction of this uncommon modification from amino acid precursors are unknown. Synthetic piperazinone-containing molecules have diverse antimicrobial, antiviral, anticancer, and anti-inflammatory bioactivity profiles, and determining biosynthetic routes for the assembly of this heterocycle may enhance drug discovery and medicinal chemistry efforts. We identified a putative TLN-05220 biosynthetic gene cluster (BGC) in the commercially available strain M. echinospora ATCC 15837 containing both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes (tln1 and tln5) that putatively encode pyridoxal 5-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), identified L-alanine, L-serine, and glycine as metabolic precursors of TLN-05220. Subsequent in vitro enzymology established that Tln1 is a PLP-dependent alanine racemase, while Tln5 performs a stereoselective {beta}-substitution reaction of O-phospho-L-serine with a preferential D-alanine nucleophile. Alanine racemization and Tln5 pseudodipeptide L-serine-C{beta}-N-D-alanine (D,L-PDP) incorporation into TLN-05220 were further supported using deuterated intermediates and mass spectrometry techniques. Establishing the enzymes that catalyze amino acid installation within TLN-05220 inspires further biosynthetic discovery and engineering while enabling the biocatalytic syntheses of novel amino acid-containing polyketide antibiotics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/685158v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@4402e8org.highwire.dtl.DTLVardef@11af789org.highwire.dtl.DTLVardef@1717c5borg.highwire.dtl.DTLVardef@19b6529_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Separation of halide oxidation and substrate halogenation chemistries rationalizes site-selective vanadium dependent haloperoxidase catalysis

Vanadium-dependent haloperoxidases (VHPOs) catalyze the halogenation of organic molecules under mild aqueous conditions. Selective bacterial VHPOs exhibit exquisite regio- and enantiocontrol, however the precise mechanisms dictating selectivity have remained elusive. We have solved the single-particle cryo-electron microscopy (cryo-EM) structure of a selective bromoperoxidase from Enhygromyxa salina (esVHPO). Mutagenesis demonstrates that halide oxidation and substrate halogenation occur in two distinct pockets, with halide transfer mediated by critical lysine residue K329. Isolation of a stable intermediate following bromide oxidation (BrOx) enables single turnover catalysis in the presence of organic substrate; subsequent application of a chemoselective fluorescent probe provides support for an intermediate bromamine involved in selectivity. Cryo-EM of the BrOx state reveals a camera shutter mechanism that compacts the halide entry tunnel and vanadate pocket, minimizing the premature dissociation of hypohalous acid. These findings collectively unveil a multilayered halogen trapping and transfer mechanism and provide a rationale for selective VHPO catalysis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/684477v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@6154b9org.highwire.dtl.DTLVardef@169fdorg.highwire.dtl.DTLVardef@d56940org.highwire.dtl.DTLVardef@f11028_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

IDBac: an open-access web platform and compendium for the identification of bacteria by MALDI-TOF mass spectrometry.

The identification and analysis of bacteria is central to the microbiological sciences. While gene sequencing methods have been the standard to achieve this, use of MALDI-TOF mass spectrometry (MS), particularly in clinical microbiology, provides high-throughput identification to the subspecies level. However, biotyping has yet to be adopted outside of clinical settings due to the lack of a centralized public database of MS protein signatures that would facilitate isolate identification via spectral comparison. Further, current platforms lack meaningful ways to compare multiple properties from large numbers of bacterial isolates. Herein we present the IDBac web platform, a crowd-sourced central knowledgebase of protein MS signatures of >1400 strains spanning 6 bacterial phyla. Accompanying the knowledgebase is analysis infrastructure to identify unknown isolates, probe relationships within culture collections using metadata integration, and visualize specialized metabolite differences within groups of closely related bacteria. To highlight this utility and encourage wide community contribution, examples of each are presented.

microbiology↗

bioRxiv2025

CURE-ating the substrate scope and functional residues of nonheme iron(II) α-ketoglutarate dependent hydroxylase BesE

Nonheme iron (II) -ketoglutarate-dependent dioxygenases (Fe/KGs) play important roles in functionalizing biological substrates from individual amino acids up to macromolecules. BesE, a unique homolog from the actinobacterial {beta}-ethynylserine biosynthetic pathway, catalyzes a highly selective hydroxylation on dipeptide substrate {gamma}-L-glutamyl-L-propargylglycine (1). Inspired by this transformation, our year-long Course-based Undergraduate Research Experience (CURE) laboratory interrogated BesE catalysis using an interdisciplinary approach. After establishing a modular chemical synthesis of 1, we rationally designed fourteen non-native analogs with key alterations to specific substrate moieties putatively involved in enzymatic recognition. Following in vitro enzymology and the application of chemical derivatization techniques compatible with all substrate analogs and putative products, we determined that the terminal alkyne moiety is not essential while reinforcing the significance of the {gamma}-glutamyl moiety for hydroxylation activity. Thirteen rationally designed BesE mutants established the importance of polar active site residues for substrate recognition and catalysis. This work establishes a baseline of BesE recognition from both a chemical and biochemical perspective and contributes to the growing understanding about Fe/KG recognition on biomedically relevant targets. Moreover, this contributes to the growing examples of using natural products and their biosynthetic enzymology as a vibrant platform for the interdisciplinary training of early career biomedical researchers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=53 SRC="FIGDIR/small/681649v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1d6f5aaorg.highwire.dtl.DTLVardef@19aa649org.highwire.dtl.DTLVardef@1b2f8dorg.highwire.dtl.DTLVardef@cf0867_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Development of a High-Throughput Ion Mobility Spectrometry-Based Screening Platform for Kainoid Synthase Biocatalysts

Random mutagenesis generates large, diverse enzyme libraries, however high-throughput screening is challenging when transformations yield isomeric small molecule products. We developed a matrix-assisted laser desorption/ionization-trapped ion mobility spectrometry-mass spectrometry (MALDI-TIMS-MS) platform for rapid, label-free screening of libraries of kainoid synthases, Fe/KG-dependent cyclases that produce the isomeric neurotransmitters kainic acid (KA) and kainic acid lactone (KAL). This platform achieves near-baseline separation of KA and KAL directly from microbial biomass, with parallel reaction monitoring-PASEF (PRM-PASEF) enhancing signal-to-noise for isomeric product ratio analysis. Using this workflow, we screened a 318-variant DsKabC/GfKabC DNA shuffle library in under 30 minutes, identifying seven variants with improved KAL conversion. Two variants showed a near-complete shift toward KAL production with greater substrate consumption, while retaining favorable expression profiles compared to GfKabC. These results establish MALDI-TIMS-MS as a generalizable platform for high-throughput isomeric product screening and provides new kainoid synthase variants to probe structure-function relationships for further development as biocatalysts. Importantly, this screening approach can be broadly applied to interrogate enzyme variants acting on small molecules, delivering rapid and accurate quantitative insights without reliance on chromatography, enzyme purification, or substrate derivatization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/674781v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@185877dorg.highwire.dtl.DTLVardef@18234d6org.highwire.dtl.DTLVardef@15b0df9org.highwire.dtl.DTLVardef@f28f8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Vanadium-dependent haloperoxidases from diverse microbes halogenate exogenous alkyl quinolone quorum sensing signals

Site-selective vanadium-dependent haloperoxidases (VHPOs) are a unique enzyme family that catalyze selective halogenation reactions previously characterized within bacterial natural product biosynthetic pathways. However, the broader chemical roles and biological distribution of these halogenases remains to be explored. Using bioinformatic methods, we have defined a VHPO subfamily that regioselectively brominates alkyl quinolone (AQ) quorum sensing molecules. In vitro AQ halogenation activity was demonstrated from phylogenetically distinct bacteria lacking established AQ biosynthetic pathways and sourced from diverse environments. AQ-VHPOs show high sequence and biochemical similarities with negligible genomic synteny or biosynthetic gene cluster co-localization. Exposure of VHPO-containing microbes to synthetic AQs or established bacterial producers identifies the chemical and spatial response to subvert their bacteriostatic effects. The characterization of novel homologs from bacterial taxa without previously demonstrated vanadium enzymology suggests VHPO-mediated AQ bromination is a niche to manipulate the chemical ecology of microbial communities. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/606109v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@17a6b2aorg.highwire.dtl.DTLVardef@119f77org.highwire.dtl.DTLVardef@fc5855org.highwire.dtl.DTLVardef@10b6e66_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisA broadly distributed subfamily of bacterial site-selective VHPOs provides protection against exogenously encountered bacteriostatic alkyl quinolone quorum sensing signals.

biochemistry↗

bioRxiv2023

Algal kainoid synthases exhibit substrate-dependent hydroxylation and cyclization activities

FeII/-ketoglutarate-dependent dioxygenases (Fe/KG) are a large enzyme family that functionalize C-H bonds on diverse organic substrates. Although Fe/KG homologs catalyze an array of chemically useful reactions, hydroxylation typically predominates. Microalgal DabC uniquely forms a novel C-C bond to construct the bioactive pyrrolidine ring in domoic acid biosynthesis. However, this kainoid synthase exclusively performs a stereospecific hydroxylation reaction on its cis substrate regioisomer. Mechanistic and kinetic analyses with native and alternative substrates identified a 20-fold rate increase in DabC radical cyclization over {beta}-hydroxylation, with no observable 1,5-hydrogen atom transfer. Moreover, this dual activity was conserved among macroalgal RadC1 and KabC homologs and provided insight into substrate recognition and reactivity trends. Investigation of this substrate-dependent chemistry improves our understanding of Fe/KG enzymes and their biocatalytic application.

biochemistry↗

bioRxiv2023

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

Cyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cya-nobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA L-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively {gamma}-hydroxylated L-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate L-enduracididine biosynthesis from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using spectroscopic, stable isotope labeling techniques, and X-ray crystal structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the - and {beta}-positions of its substrate prior to catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate bound GntC structures and activity assays on sitespecific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enables an improved understanding of how Nature divergently produces cyclic arginine ncAAs and generates additional tools for their biocatalytic production and downstream biological applications.

biochemistry↗

bioRxiv2020

The Phaeodactylum tricornutum Diaminopimelate Decarboxylase was Acquired via Horizontal Gene Transfer from Bacteria and Displays Substrate Promiscuity

Diatoms are predicted to synthesize certain amino acids within the chloroplast, including L-lysine via a diaminopimelate-dependent pathway. Herein, we report that the model diatom, Phaeodactylum tricornutum, possesses a chimeric lysine biosynthetic pathway, which coalesces bacterial and plant genes, and is terminated by a chloroplast-localized diaminopimelate decarboxylase (DAPDC, PtLYSA). We show that while RNAi ablation of PtLYSA is either synthetically lethal or concomitant with a slower growth rate, Cas9-mediated mutagenesis of PtLYSA results in recovery of heterozygous cells lines, suggesting that PtLYSA is an essential gene. Previously characterized DAPDCs are unique within the PLP-dependent decarboxylases where catalysis occurs at the D-stereocenter of the substrate and display a strict stereochemical preference for a (D,L)- or meso-substrate and not the D,D- or L,L-isomers of diaminopimelate (DAP) to synthesize L-lysine. Using decarboxylation assays and differential scanning calorimetry analyses, we validate that PtLYSA is a bona fide DAPDC and uncover its unexpected stereopromiscuous behavior in substrate specificity. The crystal structure of PtLYSA confirms the enzyme is an obligate homodimer in which both protomers reciprocally participate in the active site. The structure underscores features unique to the PtLYSA clan of DAPDC and provides structural insight into the determinants responsible for the substrate-promiscuity observed in PtLYSA.

biochemistry↗

bioRxiv2020

Algal Neurotoxin Biosynthesis Repurposes the Terpene Cyclase Structural Fold Into an N-prenyltransferase

Prenylation is a common biological reaction in all domains of life whereupon prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these biotransformations are structurally distinct from ubiquitous terpene cyclases that instead assemble terpene molecules via intramolecular rearrangements. Herein we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical Mg2+-binding motif. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to alter their isoprene donor specificities (geranyl versus dimethylallyl diphosphate) by a single amino acid switch. While the diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor. SignificanceDomoic acid is a neurotoxin produced by marine algae that readily bioaccumulates in shellfish and significantly impacts both human and animal life. The first committed step of the biosynthesis of domoic acid is the N-prenylation of L-glutamic acid by the enzyme DabA. By solving the crystal structure of DabA, we demonstrate that this enzyme has repurposed the common terpene cyclase fold to catalyze an extremely unusual reaction, N-prenylation of an unactivated primary amine. Application of these structural insights enabled rational engineering of two N-prenyltransferase enzymes to accept alternative prenyl donors. Ultimately, these results not only expand the scope of reactions catalyzed by a terpene cyclase family member, but will help inform future domoic acid environmental monitoring efforts.

biochemistry↗