Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater
 oxydans

Yin, Biaolin; Cui, Dongbing; Zhang, Lujia; Jiang, Shuiqin; Machida, Satoru; Yuan, Yusheng; Wei, Dongzhi

doi:10.1002/prot.24603

Cited by 6 publications

(4 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Seven out of nine had an Ala at position 100 (the residue number of Km CR). In contrast, Asn was in SDR Gox2253 (PDB: 3WJ7) (Figure 2C), which was still consistent with the “A/N‐S‐Y‐K” catalytic tetrad theory (Yin et al, 2014). In another exception, Thr was observed in a vestitone reductase (PDB: 2P4H) at position 100, which was consistent with the predicted catalytic residues in NCBI (GenBank Q4316.1) (Shao et al, 2007).…”

Section: Resultssupporting

confidence: 79%

Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase

Cheng

Zhai

Gao

et al. 2021

Biotech & Bioengineering

View full text Add to dashboard Cite

Enzyme engineering toward catalytic-tetrad residues usually results in activity loss. Unexpectedly, we found that a directed evolution campaign yielded a beneficial residue A100 in KmCR (a carbonyl reductase from Kluyveromyces marxianus ZJB14056), which is a residue of catalytic tetrad and conserved according to multiple sequence alignment. Inspired by this finding, we performed saturation mutagenesis on all the four residues of catalytic tetrad of KmCR. A number of variants with improved enzymatic activities were obtained. Among them, the variant KmCR_A100S exhibited increased catalytic efficiency (k cat /K M = 47.3 s −1 •mM −1 ), improved stereoselectivity (from moderate selectivity (de P = 66.7%) to strict (S)-selectivity (de P > 99.5%)), and extended substrate scope, compared to those of KmCR_WT. In silico analysis showed that a relay system was rebuilt in KmCR via the beneficial residue S100. Furthermore, comparison of 11 protein engineering campaigns indicated that the beneficial position is easily overlooked due to the long distance (>10 Å) from ketone substrates. Since CRs share similar catalytic mechanism, the knowledge gained from this study has universal significance to CR engineering.

show abstract

Section: Resultssupporting

confidence: 79%

Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase

Cheng

Zhai

Gao

et al. 2021

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…A search for the closest structural homologues to 3β-HSD2 retrieved the structure of the short-chain dehydrogenase/reductase protein Gox2253 from Gluconobacter oxydans as the best template for homology modeling [13]. The alignment used for homology modeling spans residues 1-358 of 3β-HSD2 and is reported in figure 3a.…”

Section: Resultsmentioning

confidence: 99%

“…The short-chain dehydrogenase Gox2253 from Gluconobacter oxydans was identified as the best homologue of known structure (pdbcode: 3WJ7) [13]. The alignment produced by Delta-Blast was used as input to construct and energy-minimize the homology model, using Modeller version 9.13 [14].…”

Section: Methodsmentioning

confidence: 99%

A New Homozygous Frameshift Mutation in the HSD3B2 Gene in an Apparently Nonconsanguineous Italian Family

et al. 2016

View full text Add to dashboard Cite

Background: 3β-Hydroxysteroid dehydrogenase (3β-HSD) deficiency is a rare cause of congenital adrenal hyperplasia (CAH) caused by inactivating mutations in the HSD3B2 gene. Patient and Methods: We report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child born to apparently nonconsanguineous parents and presenting ambiguous genitalia and salt wasting. The steroid profile showed elevated concentrations of 17-hydroxyprogesterone, androstenedione, ACTH and plasma renin, but normal values of cortisol and dehydroepiandrosterone sulfate. Unexpectedly, plasma aldosterone was high. For structural and functional analyses, the three-dimensional structure of 3β-HSD2 was modeled using the crystal structure of the short-chain dehydrogenase Gox2253 from Gluconobacter oxydans as a template. Results: The direct DNA sequence of the child revealed a new homozygous frameshift mutation in exon 4 of the HSD3B2 gene, a single nucleotide deletion at codon 319 [GTC(Val)→GC], yielding premature stop codon in position 367. Molecular homology modeling and secondary structure predictions suggested that the variant sequence might both alter the substrate-binding cleft and compromise the overall stability of the enzyme. Conclusion: We have described the first HSD3B2 gene mutation in the Italian population and analyzed its effect in the context of the 3β-HSD2 structure and function.

show abstract

“…To engineer Sm SDR with higher activity and improve this biotransformation process, a detailed structure-function analysis of Sm SDR and Sm SDR-substrate interactions is necessary. In addition, the SDR proteins have been used to alter coenzyme and substrate specificity for biological application 26 , 27 .…”

Section: Introductionmentioning

confidence: 99%

Structure-guided design of Serratia marcescens short-chain dehydrogenase/reductase for stereoselective synthesis of (R)-phenylephrine

Liu

Kuan

Tsou

et al. 2018

Sci Rep

View full text Add to dashboard Cite

Bioconversion is useful to produce optically pure enantiomers in the pharmaceutical industry, thereby avoiding problems with side reactions during organic synthesis processes. A short-chain dehydrogenase/reductase from Serratia marcescens BCRC 10948 (SmSDR) can stereoselectively convert 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) into (R)-phenylephrine [(R)-PE], which is marketed medically as a nasal decongestant agent. The whole-cell conversion process for the synthesis of (R)-PE using SmSDR was reported to have an unexpectedly low conversion rate. We reported the crystal structure of the SmSDR and designed profitable variants to improve the enzymatic activity by structure-guided approach. Several important residues in the structure were observed to form hydrophobic clusters that stabilize the mobile loops surrounding the pocket. Of these, Phe98 and Phe202 face toward each other and connect the upper curvature from the two arms (i.e., the α7 helix and loopβ4–α4). The mutant structure of the double substitutions (F98YF202Y) exhibited a hydrogen bond between the curvatures that stabilizes the flexible arms. Site-directed mutagenesis characterization revealed that the mutations (F98Y, F98YF202Y, and F98YF202L) of the flexible loops that stabilize the region exhibited a higher transformation activity toward HPMAE. Together, our results suggest a robust structure-guided approach that can be used to generate a valuable engineered variant for pharmaceutical applications.

show abstract

Structural insights into substrate and coenzyme preference by SDR family protein Gox2253 from Gluconobater oxydans

Cited by 6 publications

References 27 publications

Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase

Tuning enzymatic properties by protein engineering toward catalytic tetrad of carbonyl reductase

A New Homozygous Frameshift Mutation in the <b><i>HSD3B2</i></b> Gene in an Apparently Nonconsanguineous Italian Family

Structure-guided design of Serratia marcescens short-chain dehydrogenase/reductase for stereoselective synthesis of (R)-phenylephrine

Contact Info

Product

Resources

About