Abstract：
:The acetoacetate decarboxylase-like superfamily (ADCSF) is a little-explored group of enzymes that may contain new biocatalysts. The low level of sequence identity (∼20%) between many ADCSF enzymes and the confirmed acetoacetate decarboxylases led us to investigate the degree of diversity in the reaction and substrate specificity of ADCSF enzymes. We have previously reported on Sbi00515, which belongs to Family V of the ADCSF and functions as an aldolase-dehydratase. Here, we more thoroughly characterize the substrate specificity of Sbi00515 and find that aromatic, unsaturated aldehydes yield lower KM and higher kcat values compared to those of other small electrophilic substrates in the condensation reaction. The roles of several active site residues were explored by site-directed mutagenesis and steady state kinetics. The lysine-glutamate catalytic dyad, conserved throughout the ADCSF, is required for catalysis. Tyrosine 252, which is unique to Sbi00515, is hypothesized to orient the incoming aldehyde in the condensation reaction. Transient state kinetics and an intermediate-bound crystal structure aid in completing a proposed mechanism for Sbi00515.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Mydy LS,Hoppe RW,Hagemann TM,Schwabacher AW,Silvaggi NRdoi
10.1021/acs.biochem.9b00652subject
Has Abstractpub_date
2019-10-08 00:00:00pages
4136-4147issue
40eissn
0006-2960issn
1520-4995journal_volume
58pub_type
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