Mechanism of a hypothiocyanous acid reductase

Western Michigan University received a $558,493 Project Grant from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training program (CFDA 93.859), awarded September 11, 2025, with a completion date of August 31, 2028. The award supports fundamental research to elucidate the chemical and catalytic mechanisms of hypothiocyanous acid reductase (HAR), an enzyme expressed by human-colonizing bacteria including major pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli O157:H7. HAR enables bacterial resistance to hypothiocyanous acid (HOSCn), a potent antimicrobial compound produced by the innate immune system in human saliva, tears, and epithelial fluids.

The research deliverables include investigation of HAR's reaction mechanisms through mutagenesis, kinetic analysis, and transient kinetics studies to identify reaction intermediates and define catalytic pathways. Specific research objectives encompass determining the roles of HAR-specific active site residues in HOSCn reduction, elucidating the sequence of events during HAR's reaction with NAD(P)H cofactors, and understanding HAR's unusual dual specificity for both NADPH and NADH as reductants. Additionally, protein engineering will generate HAR variants with altered cofactor specificity to advance understanding of HAR's catalytic mechanisms. These findings may inform development of therapeutic strategies targeting this virulence factor in antibiotic-resistant bacterial pathogens.

Abstract

The innate immune system deploys a suite of oxidants to combat infection by bacterial pathogens and control the human microbiome. Among these are hypothiocyanous acid (hoscn), a potent thiol-specific oxidant capable of killing many bacteria. Unlike other immune-derived oxidants, hoscn is non-toxic to human cells and is constantly produced in saliva, tears and epithelial fluids lining the respiratory tract as a defense against bacterial colonization. We recently discovered that many human-colonizing bacteria express a flavin-dependent hoscn reductase (HAR) that rapidly neutralizes hoscn upon reduction with NAD(p)h, providing resistance against hoscn toxicity. HAR is a member of the flavoprotein disulfide reductase (FDR) family, albeit with an unusual function, as these enzymes typically catalyze the reversible reduction of disulfide-containing substrates. HAR is also found in major human pathogens like staphylococcus aureus, streptococcus pneumoniae and escherichia coli O157:H7, for which antibiotic resistance is a growing threat, and deletion of the HAR gene dramatically attenuates s. Pneumoniae colonization in mouse models, establishing HAR as an important virulence factor. This project aims to elucidate the chemical and catalytic mechanisms of HAR. The first aim involves mutagenesis and stopped-flow kinetics to identify reaction intermediates and determine the role of HAR-specific active site residues in the mechanism of hoscn reduction by HAR. The second aim utilizes transient kinetics and measurement of kinetic isotope effects to define the sequence of events that occur during HAR's reaction with NAD(p)h. The third aim seeks to understand the mechanism behind HAR's ability to use both nadph and nadh as a reductant, which is highly unusual among FDR family enzymes. Protein engineering will be used to generate HAR variants with altered nadph/nadh specificity, and these will be used to test the hypothesis that HAR's LAX pyridine nucleotide specificity is critical for its ability to provide resistance against hoscn. Together, these research activities will generate fundamental insights into a widespread bacterial defense mechanism against the innate immune system, paving the way for developing inhibitors targeting HAR function. Additionally, this project will enrich the research environment at a regional undergraduate institution, exposing undergraduate students to meritorious research involving enzymology and transient kinetics.