Essential Tyrosine Residues in 3-Ketosteroid- '-Dehydrogenase from Rhodococcus rhodochrous

Fujii, Chifumi; Morii, Shingo; Kadode, Michiaki; Sawamoto, Shizue; Iwami, Masafumi; Itagaki, Eiji

doi:10.1093/oxfordjournals.jbchem.a022500

Cited by 18 publications

(11 citation statements)

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“…Based on chemical modification, mutagenesis, and kinetics experiments, several amino acid residues of the ⌬ 1 -KSTD from Rhodococcus rhodochrous (formerly Nocardia corallina) were identified as important for catalytic activity, including one or two histidines, an arginine, and Tyr 121 , and several other residues were shown to be important for binding of the steroid substrates, such as Tyr 104 and Tyr 116 (14,15 (2). Previously, we described the purification, crystallization, and preliminary x-ray crystallographic analysis of ⌬ 1 -KSTD1 (17).…”

Section: -Ketosteroid ⌬mentioning

confidence: 99%

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

Rohman

Oosterwijk

Thunnissen

et al. 2013

Journal of Biological Chemistry

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Background: 3-Ketosteroid ⌬ 1 -dehydrogenases catalyze the 1,2-desaturation of 3-ketosteroids. Results: First structures of the enzyme from Rhodococcus erythropolis SQ1, combined with site-directed mutagenesis, clarify its catalytic mechanism. Conclusion: Tyr 487 and Gly 491 promote keto-enol tautomerization, whereas Tyr 318 /Tyr 119 and FAD abstract a proton and hydride ion, respectively. Significance: This study is an important step toward tailoring the enzyme for steroid biotransformation applications.

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Section: -Ketosteroid ⌬mentioning

confidence: 99%

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

Rohman

Oosterwijk

Thunnissen

et al. 2013

Journal of Biological Chemistry

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“…The GenBank accession number for the sequence reported in this paper is AY078169 stimulated research on KSTD proteins and their corresponding genes from different genera and species Drobnic et al, 1993 ;Fujii et al, 1999 ;Itagaki et al, 1990 ;Kaufmann et al, 1992 ;Molna! r et al, 1995 ;Morii et al, 1998 ;Plesiat et al, 1991 ;Sih & Bennet, 1962 ;Wagner et al, 1992 ;Wovcha et al, 1979).…”

Section: Abbreviationsmentioning

confidence: 99%

Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid Δ1-dehydrogenase isoenzyme b bThe GenBank accession number for the sequence reported in this paper is AY078169

2002

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Previously, Rhodococcus erythropolis SQ1 kstD, encoding ketosteroid ∆ 1 -dehydrogenase (KSTD1) was characterized. Surprisingly, a kstD gene deletion mutant (strain RG1) grew normally on steroids. UV mutagenesis of strain RG1 allowed isolation of strains (e.g. strain RG1-UV29) unable to perform the ∆ 1 -dehydrogenation of 4-androstene-3,17-dione (AD) and 9α-hydroxy-4-androstene-3,17-dione (9OHAD). Functional complementation of strain RG1-UV29 with total genomic DNA of strain RG1 resulted in identification of a 1698 nt ORF (kstD2) showing clear similarity (35 % identity at amino acid sequence level) with KSTD1. Expression of kstD2 in Escherichia coli resulted in high KSTD2 activity levels. Single gene deletion mutants of either kstD (strain RG1) or kstD2 (strain RG7) appeared unaffected in growth on the steroid substrates AD, 1,4-androstadiene-3,17-dione and 9OHAD. Strain RG7, but not strain RG1, showed temporary accumulation of 9OHAD during AD conversion. A kstD kstD2 double deletion mutant (strain RG8) was constructed. Strain RG8 was unable to grow on steroid substrates, had undetectable steroid ∆ 1 -dehydrogenation activity and efficiently converted AD into 9OHAD. Strain SQ1 thus employs two KSTD isoenzymes in steroid catabolism. Analysis of two null mutants in KSTD2 showed that the semi-conserved Ser325 and the highly conserved Thr503 play a role in KSTD enzyme activity.

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“…One of the most important and well-studied enzymes in microbial sterol metabolism is D-1,2 steroid dehydrogenase, which introduces a 1,2 double bond in the Aring of the steroidal molecule [5]. The enzyme has been described in Cylindrocarpo radicicola, P. testosteroni, Mycobacterium sp.…”

Section: Discussionmentioning

confidence: 99%

Resistance to androstanes as an approach for androstandienedione yield enhancement in industrial mycobacteria

Pérez¹,

Falero²,

Llanes³

et al. 2003

Journal of Industrial Microbiology and Biotechnology

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The resistance to androstandienedione (ADD) of industrial mycobacteria was demonstrated as a valuable approach to increasing ADD yield in sterol fermentations. Colonies growing at 1 mg/ml ADD in culture medium after nitrosoguanidine mutagenesis showed a differential behavior in respect to parentals in cholesterol biotransformation. In the presence of exogenous ADD, a substantial depletion of ADD production was observed in parental strains B3683 and Ex4, whereas it was unaffected, and even increased, in resistant colonies. An apparent reduction from ADD to androstandione and testosterone was also noticed. Furthermore, the ADD resistance phenotype may be related to the increase in steroid 1,2 dehydrogenase activity.

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Essential Tyrosine Residues in 3-Ketosteroid- '-Dehydrogenase from Rhodococcus rhodochrous

Cited by 18 publications

References 0 publications

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

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

Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid Δ1-dehydrogenase isoenzyme b bThe GenBank accession number for the sequence reported in this paper is AY078169

Resistance to androstanes as an approach for androstandienedione yield enhancement in industrial mycobacteria

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