Biology subjects

Howe, G. W.

Publications and source records attributed to Howe, G. W..

4 recordsLinked to original sources

bioRxiv2025

Characterization of a novel fatty acid-modifying pathway toward the biosynthesis of tambjamine BE-18591 in Streptomyces

Tambjamines are a class of bacterial bipyrrolic natural products with potent biological activity. Recently, the first Actinomycete biosynthetic gene cluster (BGC) responsible for the production of a tambjamine (BE-18591) was discovered in Streptomyces albus, and bioinformatic analysis suggested the alkylamine tail component is constructed using disparate biosynthetic logic than employed by Proteobacteria to assemble structurally similar tambjamines YP1 and MYP1. Here, we report the experimental characterization of four novel streptomycete proteins and demonstrate that these enable the unique, late-stage assembly of the alkylamine component of BE-18951. Specifically, a fatty acyl-carrier protein (TabQ) is loaded with a 12-carbon acyl chain, selected for, in part, through the action of an editing type II thioesterase (TabJ). The resulting C12-TabQ adduct is then processed to an aldehyde by a novel acyl-ACP reductase (TabE) that harbors none of the telltale amino acid signatures that typically identify these proteins. The resulting aldehyde is finally converted to the amine by an{omega} -transaminase (TabA) that demonstrates some degree of promiscuity. These four proteins encoded by the BE-18591 BGC in Streptomyces albus enable the assembly of the fatty amine that is ultimately incorporated into the tambjamine. Our findings highlight the disparate chemical logic employed by Proteo- and Actinobacteria for the biosynthesis of the alkylamine components of tambjamine natural products.

biochemistry↗

bioRxiv2025

Characterization of bacterial fucokinase/GDP-fucose pyrophosphorylase (FKP) enzymes supports the evolution of interdomain communication and modularity in the FKP family

L-fucokinase (FUK) and GDP-fucose pyrophosphorylase (GFPP) salvage free L-fucose and synthesize the valuable nucleotide-sugar GDP-L-fucose (GDP-Fuc). Some organisms express these enzymes as one bifunctional polypeptide called L-fucokinase/GDP-fucose pyrophosphorylase (FKP), which has attracted attention for use in the chemoenzymatic synthesis of GDP-Fuc. Despite the documented use of the FKP from Bacteroides fragilis (BfFKP), the evolutionary origins of these enzymes and their relationships to monofunctional FUKs and GFPPs are poorly understood. We hypothesized that biochemical characterization of these proteins coupled with an evolutionary analysis would uncover the natural diversity of FKPs, facilitating the discovery of new biocatalysts. Phylogenetic and sequence similarity network (SSN) analyses distinguished FKPs from FUKs and GFPPs, suggesting that FKPs originate from one ancestral fusion event between these domains. To evaluate how environmental factors might select for functional diversity within the FKP family, we recombinantly expressed and purified a putative FKP from the thermophilic bacterium Thermophagus xiamenensis (TxFKP). This enzyme exhibited in vitro kinase and pyrophosphorylase activities and demonstrated subtle kinetic differences compared to BfFKP. While alanine scanning mutational analysis of the TxFKP FUK and GFPP domains supported the role of conserved residues that TxFKP uses to coordinate substrate binding and catalysis, other mutations in the TxFKP GFPP domain influenced kinase activity differentially for the substrates L-fucose and D-arabinose, showing an unprecedented role for the GFPP domain in FUK substrate specificity. Finally, thermal shift profiles of TxFKP and BfFKP were biphasic and provided new insights into how these enzymes have evolved to respond to different sugar substrates.

biochemistry↗

bioRxiv2024

Genome mining leads to the identification of a stable and promiscuous Baeyer-Villiger monooxygenase from a thermophilic microorganism

Baeyer-Villiger monooxygenases are NAD(P)H-dependent flavoproteins that catalyze oxygen insertion reactions which convert ketones to valuable esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel Baeyer-Villiger monooxygenases that are both stable and substrate promiscuous. Our genome mining efforts led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature recorded to date for a naturally sourced Baeyer-Villiger monooxygenase, ii) a remarkable kinetic stability across a wide range of conditions, and iii) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Kinetic characterization of this enzyme was undertaken to identify the optimal conditions for ssnBVMO catalysis, and a subsequent quantitative assay using propiophenone as a substrate afforded more than 95% conversion. To spur the implementation of this enzyme as an oxidative biocatalyst, several fusion proteins were constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These self-sufficient enzymes can recycle NADPH and permit oxidations to be run with sub-stoichiometric quantities of this expensive cofactor. Extensive characterization of these fusion enzymes permitted identification of PTDH-L1-ssnBVMO as the most promising oxidative biocatalyst. Results described herein demonstrate that this new monooxygenase has significant potential as a useful industrial biocatalyst for Baeyer-Villiger oxidations.

biochemistry↗

bioRxiv2024

Taxonomic distribution of SbmA/BacA and BacA-like antimicrobial peptide transporters suggests independent recruitment and convergent evolution in host-microbe interactions

Small, antimicrobial peptides are often produced by eukaryotes to control bacterial populations in both pathogenic and mutualistic symbioses. These include proline-rich mammalian immune peptides and cysteine-rich peptides produced by legume plants in symbiosis with rhizobia. The fitness of the bacterial partner is dependent upon their ability to persist in the presence of these antimicrobial peptides. In the case of Escherichia coli and Mycobacterium tuberculosis pathogens and nitrogen-fixing legume symbionts (rhizobia), the ability to survive exposure to these peptides depends on peptide transporters called SbmA (also known as BacA) or BclA (for BacA-like). However, how broadly these transporters are distributed amongst bacteria, and their evolutionary history, is poorly understood. Here, we used hidden Markov models, phylogenetic analysis, and sequence similarity networks to examine the distribution of SbmA/BacA and BclA proteins across a representative set of 1,255 species from across the domain Bacteria. We identified a total of 71 and 177 SbmA/BacA and BclA proteins, respectively. Phylogenetic and sequence similarity analyses suggest that these protein families likely did not evolve from a common ancestor and that their functional similarity is instead a result of convergent evolution. In vitro sensitivity assays using the legume peptide NCR247 and several of the newly-identified BclA proteins confirmed that transport of antimicrobial peptides is a common feature of this protein family. Analysis of the taxonomic distribution of these proteins showed that SbmA/BacA orthologs were encoded only by species in the phylum Pseudomonadota and that they were primarily identified in just two orders: Hyphomicrobiales (class Alphaproteobacteria) and Enterobacterales (class Gammaproteobacteria). BclA orthologs were somewhat more broadly distributed and were found in clusters across four phyla. These included several orders of the phyla Pseudomonadota and Cyanobacteriota, as well as the order Mycobacteriales (phylum Actinomycetota) and the class Negativicutes (phylum Bacillota). Notably, many of the clades enriched for species encoding BacA or BclA orthologs also include many species known to interact with eukaryotic hosts in mutualistic or pathogenic interactions. Collectively, these observations suggest that SbmA/BacA and BclA proteins have been repeatedly co-opted to facilitate both mutualistic and pathogenic associations with eukaryotic hosts by allowing bacteria to cope with host-encoded antimicrobial peptides.

microbiology↗