Tungstoenzymes: Occurrence, Catalytic Diversity and Cofactor Synthesis

Tungsten is the heaviest element used in biological systems. It occurs in the active sites of several bacterial or archaeal enzymes and is ligated to an organic cofactor (metallopterin or metal binding pterin; MPT) which is referred to as tungsten cofactor (Wco). Wco-containing enzymes are found in the dimethyl sulfoxide reductase (DMSOR) and the aldehyde:ferredoxin oxidoreductase (AOR) families of MPT-containing enzymes. Some depend on Wco, such as aldehyde oxidoreductases (AORs), class II benzoyl-CoA reductases (BCRs) and acetylene hydratases (AHs), whereas others may incorporate either Wco or molybdenum cofactor (Moco), such as formate dehydrogenases, formylmethanofuran dehydrogenases or nitrate reductases. The obligately tungsten-dependent enzymes catalyze rather unusual reactions such as ones with extremely low-potential electron transfers (AOR, BCR) or an unusual hydration reaction (AH). In recent years, insights into the structure and function of many tungstoenzymes have been obtained. Though specific and unspecific ABC transporter uptake systems have been described for tungstate and molybdate, only little is known about further discriminative steps in Moco and Wco biosynthesis. In bacteria producing Moco- and Wco-containing enzymes simultaneously, paralogous isoforms of the metal insertase MoeA may be specifically involved in the molybdenum- and tungsten-insertion into MPT, and in targeting Moco or Wco to their respective apo-enzymes. Wco-containing enzymes are of emerging biotechnological interest for a number of applications such as the biocatalytic reduction of CO2, carboxylic acids and aromatic compounds, or the conversion of acetylene to acetaldehyde.

Pterin function in bacteria

Pterins are widely conserved biomolecules that play essential roles in diverse organisms. First described as enzymatic cofactors in eukaryotic systems, bacterial pterins were discovered in cyanobacteria soon after. Several pterin structures unique to bacteria have been described, with conjugation to glycosides and nucleotides commonly observed. Despite this significant structural diversity, relatively few biological functions have been elucidated. Molybdopterin, the best studied bacterial pterin, plays an essential role in the function of the Moco cofactor. Moco is an essential component of molybdoenzymes such as sulfite oxidase, nitrate reductase, and dimethyl sulfoxide reductase, all of which play important roles in bacterial metabolism and global nutrient cycles. Outside of the molybdoenzymes, pterin cofactors play important roles in bacterial cyanide utilization and aromatic amino acid metabolism. Less is known about the roles of pterins in nonenzymatic processes. Cyanobacterial pterins have been implicated in phenotypes related to UV protection and phototaxis. Research describing the pterin-mediated control of cyclic nucleotide metabolism, and their influence on virulence and attachment, points to a possible role for pterins in regulation of bacterial behavior. In this review, we describe the variety of pterin functions in bacteria, compare and contrast structural and mechanistic differences, and illuminate promising avenues of future research.

Structure and Function of the Unusual Tungsten Enzymes Acetylene Hydratase and Class II Benzoyl-Coenzyme A Reductase

In biology, tungsten (W) is exclusively found in microbial enzymes bound to a bis<i>-</i>pyranopterin cofactor (bis-WPT). Previously known W enzymes catalyze redox oxo/hydroxyl transfer reactions by directly coordinating their substrates or products to the metal. They comprise the W-containing formate/formylmethanofuran dehydrogenases belonging to the dimethyl sulfoxide reductase (DMSOR) family and the aldehyde:ferredoxin oxidoreductase (AOR) families, which form a separate enzyme family within the Mo/W enzymes. In the last decade, initial insights into the structure and function of two unprecedented W enzymes were obtained: the acetaldehyde forming acetylene hydratase (ACH) belongs to the DMSOR and the class II benzoyl-coenzyme A (CoA) reductase (BCR) to the AOR family. The latter catalyzes the reductive dearomatization of benzoyl-CoA to a cyclic diene. Both are key enzymes in the degradation of acetylene (ACH) or aromatic compounds (BCR) in strictly anaerobic bacteria. They are unusual in either catalyzing a nonredox reaction (ACH) or a redox reaction without coordinating the substrate or product to the metal (BCR). In organic chemical synthesis, analogous reactions require totally nonphysiological conditions depending on Hg²⁺ (acetylene hydration) or alkali metals (benzene ring reduction). The structural insights obtained pave the way for biological or biomimetic approaches to basic reactions in organic chemistry.

Characterization of a P1-Like Bacteriophage Carrying an SHV-2 Extended-Spectrum β-Lactamase from an Escherichia coli Strain

ABSTRACTP1 bacteriophages lysogenize bacteria as independent plasmid-like elements. We describe here a P1-like bacteriophage, RCS47, carrying ablaSHV-2gene, isolated from a clinical strain ofEscherichia colifrom phylogroup B1, and we report the prevalence of P1-like prophages in naturalE. coliisolates. We found that 70% of the sequence of RCS47, a 115-kb circular molecule, was common to the reference P1 bacteriophage under GenBank accession no.AF234172.1, with the shared sequences being 99% identical. RCS47 had acquired two main foreign DNA fragments: a 9,636-bp fragment mobilized by two IS26elements containing ablaSHV-2gene, and an 8,544-bp fragment mobilized by two IS5elements containing an operon encoding a dimethyl sulfoxide reductase. The reference P1 prophage plasmid replication gene belonged to the IncY incompatibility group, whereas that of RCS47 was from an unknown group. The lytic capacity of RCS47 andblaSHV-2gene transduction, through the lysogenization of RCS47 in the recipientE. colistrains, were not demonstrated. The prevalence of P1-like prophages in various animal and humanE. colistrain collections, as determined by the PCR detection ofrepL, the lytic replication gene, was 12.6%. No differences in the prevalences of these prophages were found between extended-spectrum β-lactamase (ESBL)-producing and non-ESBL-producing strains (P= 0.69), but this prevalence was lower in phylogroup B2 than in the other phylogroups (P= 0.008), suggesting epistatic interactions between P1 family phages and the genetic background ofE. colistrains. P1-like phages are part of the mobile elements that carry antibiotic resistance. The high prevalence of P1-like prophages suggests their role may be underestimated.