Abstract

The gene encoding an NAD+-dependent, 3-hydroxyisobutyrate dehydrogenase (3HIBDH-IV) from Pseudomonas denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL 21 (DE3) and characterized to understand its physiological relevance in the degradation of 3-hydroxypropionic acid (3-HP). The deduced amino acid sequence showed high similarity to other 3-hydroxyisobutyrate dehydrogenase isozymes (3HIBDHs) of P. denitrificans ATCC 13867. A comparison of 3HIBDH-IV with its relevant enzymes along with molecular docking studies suggested that Lys171, Asn175 and Gly123 are important for its catalytic function on 3-hydroxyacids. The recombinant 3HIBDH-IV was purified to homogeneity utilizing a Ni-NTA-HP resin column in high yield. 3HIBDH-IV was very specific to (S)-3-hydroxyisobutyrate, but also catalyzed the oxidation of 3-HP to malonate semialdehyde. The specific activity and half-saturation constant (K m) for 3-HP at 30°C and pH 9.0 were determined to be 17 U/mg protein and 1.0 mM, respectively. Heavy metals, such as Ag+ and Hg2+, completely inhibited the 3HIBDH-IV activity, whereas dithiothreitol, 2-mercaptoethanol and ethylenediaminetetraacetic acid increased its activity 1.5–1.8-fold. This paper reports the characteristics of 3HIBDH-IV as well as its probable role in 3-HP degradation.

Highlights

  • Several strains of Escherichia coli were recently developed to produce the commercially important chemical, 3-hydroxypropionic acid (3-HP), from glycerol [1,2]

  • 3-HP can be degraded via two routes: 1) malonate semialdehyde, which is generated by 3hydroxypropionate dehydrogenase (EC: 1.1.1.59), and 2) 3hydroxypropionyl-CoA, which is produced by either 3-hydroxypropionyl-CoA synthetase (EC: 6.2.1.36) or 3-hydroxyisobutyryl-CoA hydrolase (EC: 3.1.2.4) [427]

  • In an effort to develop an efficient P. denitrificans for coenzyme B12-free 3-HP production, we examined the enzymatic degradation of 3-HP

Read more

Summary

Introduction

Several strains of Escherichia coli were recently developed to produce the commercially important chemical, 3-hydroxypropionic acid (3-HP), from glycerol [1,2]. The final titer was high (,39 g/L), the requirement of an exogenous supply of high-cost coenzyme B12 by one essential enzyme, glycerol dehydratase, was the major obstacle to the use of E. coli in the commercial production of 3-HP. 3-HP can be degraded via two routes: 1) malonate semialdehyde, which is generated by 3hydroxypropionate dehydrogenase (EC: 1.1.1.59), and 2) 3hydroxypropionyl-CoA, which is produced by either 3-hydroxypropionyl-CoA synthetase (EC: 6.2.1.36) or 3-hydroxyisobutyryl-CoA hydrolase (EC: 3.1.2.4) (www.keggpathway.com) [427]. In P. denitrificans, 3-HP degradation and enzymes, such as 3-HP dehydrogenase and 3-hydroxypropionyl-CoA synthetase or 3hydroxyisobutyryl-CoA hydrolase, have not been reported

Objectives
Methods
Results
Discussion
Conclusion

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.