3-Ketosteroid Δ¹-dehydrogenases are FAD-dependent enzymes that catalyze the 1,2-desaturation of 3-ketosteroid substrates to initiate degradation of the steroid nucleus. Here we report the 2.0 Å resolution crystal structure of the 56-kDa enzyme from Rhodococcus erythropolis SQ1 (Δ¹-KSTD1). The enzyme contains two domains: an FAD-binding domain and a catalytic domain, between which the active site is situated as evidenced by the 2.3 Å resolution structure of Δ¹-KSTD1 in complex with the reaction product 1,4-androstadiene-3,17-dione. The active site contains four key residues: Tyr¹¹⁹, Tyr³¹⁸, Tyr⁴⁸⁷, and Gly⁴⁹¹. Modeling of the substrate 4-androstene-3,17-dione at the position of the product revealed its interactions with these residues and the FAD. The C1 and C2 atoms of the substrate are at reaction distance to the N5 atom of the isoalloxazine ring of FAD and the hydroxyl group of Tyr³¹⁸, respectively, whereas the C3 carbonyl group is at hydrogen bonding distance from the hydroxyl group of Tyr⁴⁸⁷ and the backbone amide of Gly⁴⁹¹. Site-directed mutagenesis of the tyrosines to phenylalanines confirmed their importance for catalysis. The structural features and the kinetic properties of the mutants suggest a catalytic mechanism in which Tyr⁴⁸⁷ and Gly⁴⁹¹ work in tandem to promote keto-enol tautomerization and increase the acidity of the C2 hydrogen atoms of the substrate. With assistance of Tyr¹¹⁹, the general base Tyr³¹⁸ abstracts the axial β-hydrogen from C2 as a proton, whereas the FAD accepts the axial α-hydrogen from the C1 atom of the substrate as a hydride ion.

Xylan, a prominent component of cellulosic biomass, has a high potential for degradation into reducing sugars, and subsequent conversion into bioethanol. This process requires a range of xylanolytic enzymes. Among them, β-xylosidases are crucial, because they hydrolyze more glycosidic bonds than any of the other xylanolytic enzymes. They also enhance the efficiency of the process by degrading xylooligosaccharides, which are potent inhibitors of other hemicellulose-/xylan-converting enzymes. On the other hand, the β-xylosidase itself is also inhibited by monosaccharides that may be generated in high concentrations during the saccharification process. Structurally, β-xylosidases are diverse enzymes with different substrate specificities and enzyme mechanisms. Here, we review the structural diversity and catalytic mechanisms of β-xylosidases, and discuss their inhibition by monosaccharides.

3-Ketosteroid Δ1-dehydrogenase catalyzes the 1(2)-dehydrogenation of 3-ketosteroid substrates using flavin adenine dinucleotide as a cofactor. The enzyme plays a crucial role in microbial steroid degradation, both under aerobic and anaerobic conditions, by initiating the opening of the steroid nucleus. Indeed, many microorganisms are known to possess one or more 3-ketosteroid Δ1-dehydrogenases. In the pharmaceutical industry, 3-ketosteroid Δ1-dehydrogenase activity is exploited to produce Δ1-3-ketosteroids, a class of steroids that display various biological activities. Many of them are used as active pharmaceutical ingredients in drug products, or as key precursors to produce pharmaceutically important steroids. Since 3-ketosteroid Δ1-dehydrogenase activity requires electron acceptors, among other considerations, Δ1-3-ketosteroid production has been industrially implemented using whole-cell fermentation with growing or metabolically active resting cells, in which the electron acceptors are available, rather than using the isolated enzyme. In this review we discuss biotechnological applications of microbial 3-ketosteroid Δ1-dehydrogenases, covering commonly used steroid-1(2)-dehydrogenating microorganisms, the bioprocess for preparing Δ1-3-ketosteroids, genetic engineering of 3-ketosteroid Δ1-dehydrogenases and related genes for constructing new, productive industrial strains, and microbial fermentation strategies for enhancing the product yield. Furthermore, we also highlight the recent development in the use of isolated 3-ketosteroid Δ1-dehydrogenases combined with a FAD cofactor regeneration system. Finally, in a somewhat different context, we summarize the role of 3-ketosteroid Δ1-dehydrogenase in cholesterol degradation by Mycobacterium tuberculosis and other mycobacteria. Because the enzyme is essential for the pathogenicity of these organisms, it may be a potential target for drug development to combat mycobacterial infections.

3-Ketosteroid Δ¹-dehydrogenases are FAD-dependent enzymes that catalyze the introduction of a double bond between the C1 and C2 atoms of the A-ring of 3-ketosteroid substrates. These enzymes are found in a large variety of microorganisms, especially in bacteria belonging to the phylum Actinobacteria. They play a critical role in the early steps of the degradation of the steroid core. 3-Ketosteroid Δ¹-dehydrogenases are of particular interest for the etiology of some infectious diseases, for the production of starting materials for the pharmaceutical industry, and for environmental bioremediation applications. Here we summarize and discuss the biochemical and enzymological properties of these enzymes, their microbial sources, and their natural diversity. The three-dimensional structure of a 3-ketosteroid Δ¹-dehydrogenase in connection with the enzyme mechanism is highlighted.

Outbreaks of dengue virus (DENV) in Indonesia have been mainly caused by the DENV serotype-1; -2; or -3. The DENV-4 was the least-reported serotype in Indonesia during the last five decades. We recently conducted a molecular epidemiology study of dengue in the Jember regency, East Java province, Indonesia. Dengue is endemic in the region and outbreaks occur annually. We investigated the clinical characteristics and etiology of dengue-like febrile illness in this regency to understand the disease dynamics. A total of 191 patients with clinical symptoms similar to dengue were recruited during an 11-month study in 2019–2020. Children accounted for the majority of cases and dengue burden was estimated in 41.4% of the cases based on NS1 antigen, viral RNA, and IgG/IgM antibody detection with the majority (73.4%) being primary infections. Secondary infection was significantly associated with a higher risk of severe dengue manifestation. All four DENV serotypes were detected in Jember. Strikingly, we observed the predominance of DENV-4, followed by DENV-3, DENV-1, and DENV-2. Genotype determination using Envelope gene sequence revealed the classification into Genotype I, Cosmopolitan Genotype, Genotype I, and Genotype II for DENV-1, -2, -3, and -4, respectively. The predominance of DENV-4 in Jember may be associated with a new wave of DENV infections and spread in a non-immune population lacking a herd-immunity to this particular serotype.

Complete degradation of the xylan backbone of hemicellulosic plant cell walls requires the synergistic action of endo-xylanases and β-1,4-xylosidases. While endo-xylanases produce xylooligosaccharides from xylan, β-1,4-xylosidases degrade the xylooligosaccharides into xylose monomers. The glycoside hydrolase family 43 β-1,4-xylosidase from Geobacillus thermoleovorans IT-08 is a promising, heat stable catalyst for the saccharification of hemicellulosic material into simple fermentable sugars, but it is competitively inhibited by its products arabinose and xylose. As a first step to help overcome this problem, we elucidated crystal structures of the enzyme in the unliganded form and with bound products, at 1.7–2.0 Å resolution. The structures are very similar to those of other enzymes belonging to glycoside hydrolase family 43. Unexpectedly, the monosaccharides are bound in very different ways. Arabinose preferentially binds in subsite -1, while xylose exclusively interacts with subsite +1. These structures and sugar binding preferences suggest ways for improving the catalytic performance of the enzyme by rational mutational design.