Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Rohman, Ali; Oosterwijk, N. van; Thunnissen, A.M.W.H.; Dijkstra, Bauke W.

doi:10.1074/jbc.m113.522771

Cited by 55 publications

(80 citation statements)

References 39 publications

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“…ADD has been (FAD)-binding site was coincided with the sequence G-S-G-(A/G)-(A/ G)-(A/G)-X 17 -E [18,19]. According to the crystal structure of the KSDD from Rhodococcus erythropolis, the enzyme does not have any trans-membrane helices, and the protein behaves as a soluble protein [20].…”

Section: Introductionmentioning

confidence: 98%

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Zhang

Yang

et al. 2016

Electronic Journal of Biotechnology

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a b s t r a c tBackground: 3-Ketosteroid-Δ 1 -dehydrogenase (KSDD), a flavoprotein enzyme, catalyzes the bioconversion of 4-androstene-3,17-dione (AD) to androst-1,4-diene-3,17-dione (ADD). To date, there has been no report about characterization of KSDD from Mycobacterium neoaurum strains, which were usually employed to produce AD or ADD by fermentation. Results: In this work, Corynebacterium crenatum was chosen as a new host for heterologous expression of KSDD from M. neoaurum JC-12 after codon optimization of the KSDD gene. SDS-PAGE and western blotting results indicated that the recombinant C. crenatum harboring the optimized ksdd (ksdd II ) gene showed significantly improved ability to express KSDD. The expression level of KSDD was about 1.6-fold increased C. crenatum after codon optimization. After purification of the protein, we first characterized KSDD from M. neoaurum JC-12, and the results showed that the optimum temperature and pH for KSDD activity were 30°C and pH 7.0, respectively. The K m and V max values of purified KSDD were 8.91 μM and 6.43 mM/min. In this work, C. crenatum as a novel whole-cell catalyst was also employed and validated for bioconversion of AD to ADD. The highest transformation rate of AD to ADD by recombinant C. crenatum was about 83.87% after 10 h reaction time, which was more efficient than M. neoaurum JC-12 (only 3.56% at 10 h). Conclusions: In this work, basing on the codon optimization, overexpression, purification and characterization of KSDD, we constructed a novel system, the recombinant C. crenatum SYPA 5-5 expressing KSDD, to accumulate ADD from AD efficiently. This work provided new insights into strengthening sterol catabolism by overexpressing the key enzyme KSDD, for efficient ADD production.

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Section: Introductionmentioning

confidence: 98%

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Zhang

Yang

et al. 2016

Electronic Journal of Biotechnology

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“…Phylogenetic analysis leads to classify the KstD-like enzymes in at least 4 different groups, in which KstD1, KstD2, KstD3 of Rhodococcus erythropolis SQ1 are representatives of three of them [20]. The crystal structure of the enzyme KstD1 of R. erythropolis SQ1 has been elucidated [21] confirming the presence of the two domains previously described, namely a N-terminal flavin adenine dinucleotide (FAD) binding motif and a substrate-binding domain [14, 20, 22, 23]. …”

Section: Introductionmentioning

confidence: 99%

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

et al. 2017

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BackgroundThe Rhodococcus ruber strain Chol-4 genome contains at least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) that code for flavoenzymes involved in the steroid ring degradation. The aim of this work is the functional characterization of these enzymes prior to the developing of different biotechnological applications.ResultsThe three R. ruber KstD enzymes have different substrate profiles. KstD1 shows preference for 9OHAD and testosterone, followed by progesterone, deoxy corticosterone AD and, finally, 4-BNC, corticosterone and 19OHAD. KstD2 shows maximum preference for progesterone followed by 5α-Tes, DOC, AD testosterone, 4-BNC and lastly 19OHAD, corticosterone and 9OHAD. KstD3 preference is for saturated steroid substrates (5α-Tes) followed by progesterone and DOC. A preliminary attempt to model the catalytic pocket of the KstD proteins revealed some structural differences probably related to their catalytic differences. The expression of kstD genes has been studied by RT-PCR and RT-qPCR. All the kstD genes are transcribed under all the conditions assayed, although an additional induction in cholesterol and AD could be observed for kstD1 and in cholesterol for kstD3. Co-transcription of some correlative genes could be stated. The transcription initiation signals have been searched, both in silico and in vivo. Putative promoters in the intergenic regions upstream the kstD1, kstD2 and kstD3 genes were identified and probed in an apramycin-promoter-test vector, leading to the functional evidence of those R. ruber kstD promoters.ConclusionsAt least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) have been identified and functionally confirmed in R. ruber strain Chol-4. KstD1 and KstD2 display a wide range of substrate preferences regarding to well-known intermediaries of the cholesterol degradation pathway (9OHAD and AD) and other steroid compounds. KstD3 shows a narrower substrate range with a preference for saturated substrates. KstDs differences in their catalytic properties was somehow related to structural differences revealed by a preliminary structural modelling. Transcription of R. ruber kstD genes is driven from specific promoters. The three genes are constitutively transcribed, although an additional induction is observed in kstD1 and kstD3. These enzymes have a wide versatility and allow a fine tuning-up of the KstD cellular activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0657-1) contains supplementary material, which is available to authorized users.

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“…) (Rohman et al . ). The bacterial KSDDs, especially those obtained from actinobacteria such as Arthrobacter sp., Mycobacterium sp., Nocardia sp.…”

Section: Discussionmentioning

confidence: 97%

Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ¹-dehydrogenase and catalase inBacillus subtilis

et al. 2016

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Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Cited by 55 publications

References 39 publications

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ¹-dehydrogenase and catalase inBacillus subtilis

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Crystal Structure and Site-directed Mutagenesis of 3-Ketosteroid Δ1-Dehydrogenase from Rhodococcus erythropolis SQ1 Explain Its Catalytic Mechanism

Cited by 55 publications

References 39 publications

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Over-expression of Mycobacterium neoaurum 3-ketosteroid-Δ1-dehydrogenase in Corynebacterium crenatum for efficient bioconversion of 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ1-dehydrogenase and catalase inBacillus subtilis

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Efficient androst-1,4-diene-3,17-dione production by co-expressing 3-ketosteroid-Δ¹-dehydrogenase and catalase inBacillus subtilis